WO2022044702A1 - Target measurement method, device for target measurement, target measurement apparatus, and kit for target measurement - Google Patents
Target measurement method, device for target measurement, target measurement apparatus, and kit for target measurement Download PDFInfo
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- WO2022044702A1 WO2022044702A1 PCT/JP2021/028455 JP2021028455W WO2022044702A1 WO 2022044702 A1 WO2022044702 A1 WO 2022044702A1 JP 2021028455 W JP2021028455 W JP 2021028455W WO 2022044702 A1 WO2022044702 A1 WO 2022044702A1
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- target
- molecule
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- capture molecule
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2201/00—Features of devices classified in G01N21/00
- G01N2201/06—Illumination; Optics
- G01N2201/064—Stray light conditioning
Definitions
- the present invention relates to a target measurement method, a target measurement device, a target measurement device, and a target measurement kit.
- Non-Patent Documents 1 and 2 and Patent Document 1 disclose conventional measurement methods for measuring a target using a DNA microarray.
- the nucleic acid sequence measuring methods of Non-Patent Documents 1 and 2 require a washing operation for removing a target that has not been collected, and the washing operation may deteriorate the measurement accuracy. There is a problem. Further, although the nucleic acid sequence measurement method of Patent Document 1 does not require the washing operation as in Non-Patent Documents 1 and 2, the offset light emitted from the solution of the sample supplied to the DNA microarray becomes noise and is measured. There is a problem that the accuracy is deteriorated.
- the present invention has been made in view of the above circumstances, and is a target measurement method, a target measurement device, a target measurement device, and a target measurement kit that can improve the measurement accuracy of the target contained in the sample.
- the purpose is to provide.
- a target measurement method for measuring a target included in a sample The excitation light that excites the fluorescent molecule or the fluorescent molecule emits light on the solid phase surface of the substrate provided with the conjugate of the target and the capture molecule that specifically binds to the target, which is modified with the fluorescent molecule.
- the fluorescence obtained by irradiating the excitation light from the side opposite to the solid phase surface of the substrate in a state where the solution containing the absorbent substance that absorbs the fluorescence is in contact with the solid phase surface of the substrate is applied to the solid surface surface of the substrate. Is a target measurement method that measures from the opposite side.
- the target is modified with the fluorescent molecule, and the target is modified with the fluorescent molecule.
- the capture molecule is modified with the fluorescent molecule, and the capture molecule is modified with the fluorescent molecule.
- the capture molecule is modified with the fluorescent molecule, and the capture molecule is modified with the fluorescent molecule.
- the target measurement method according to [1], wherein the target is bound to the trapping molecule immobilized on the solid phase surface to obtain the conjugate.
- the target and one of the trapping molecules immobilized on the solid phase surface are supplied with the other of the target and the trapping molecule and the fluorescent molecule to obtain the conjugate.
- the target is a nucleic acid having a specific nucleic acid sequence.
- the capture molecule is a detection probe having a sequence complementary to the specific nucleic acid sequence.
- the target measurement method according to any one of [1] to [5], wherein the target is hybridized with the detection probe to obtain the conjugate.
- a target measurement device used when measuring a target contained in a sample A substrate in which either one of the target and a capture molecule specifically bound to the target is immobilized on a solid phase surface, and a substrate.
- a container to which an excitation light that excites a fluorescent molecule that modifies a bond between the target and the capture molecule or an absorbent substance that absorbs the fluorescence emitted from the fluorescent molecule is added, that is, the target and the capture molecule.
- the target measurement device wherein the container is supplied with a solution containing the capture molecule modified with the fluorescent molecule.
- the capture molecule modified with the fluorescent molecule is immobilized on the solid phase surface.
- the target measurement device wherein a solution containing the target is supplied to the container.
- Either the target or the capture molecule is immobilized on the solid phase surface.
- Measurement device [12]
- the target is a target having a specific nucleic acid sequence.
- the target measurement device according to any one of [7] to [11], wherein the capture molecule is a detection probe having a sequence complementary to the specific nucleic acid sequence.
- the target measurement device according to any one of [7] to [12], and A fluorescence reader that measures the amount of fluorescence from the target measurement device, Has a target measuring device.
- a target measurement kit used when measuring a target contained in a sample A substrate in which one of the target and a capture molecule that specifically binds to the target is immobilized on a solid phase surface, and a substrate.
- Target measurement kit including. [15] The target measurement kit according to [14], wherein the absorbent substance is preliminarily added to the container. [16] The trapping molecule is immobilized on the solid phase surface. The target measurement kit according to [14] or [15], wherein the container holds a solution containing the target modified with the fluorescent molecule and the absorbent substance. [17] The target is immobilized on the solid surface. The target measurement kit according to [14] or [15], wherein the container holds a solution containing the capture molecule modified with the fluorescent molecule and the absorbent substance.
- the capture molecule modified with the fluorescent molecule is immobilized on the solid phase surface.
- Either the target or the capture molecule is immobilized on the solid phase surface.
- the container holds a solution containing either one of the target and the capture molecule, the fluorescent molecule that binds to the conjugate of the target and the capture molecule, and the absorbance substance [14. ]
- the target is a target having a specific nucleic acid sequence.
- the target measurement method the target measurement device, the target measurement device, and the target measurement kit of the present invention, there is an effect that the measurement accuracy of the target included in the sample can be improved as compared with the conventional case.
- a method of immobilizing a DNA probe modified with a fluorescent molecule (donor fluorescent probe) and a quenching molecule (quenching probe) that specifically binds to the DNA probe on the solid phase surface of the substrate and binding the target to the donor fluorescent probe is shown. It is a figure. It is a figure which shows an example of the structure of the device for target measurement of this invention. It is a flowchart which shows an example of the operation procedure which detects a target using the device for target measurement of this invention. It is a figure which shows an example of the structure of the target measuring apparatus of this invention.
- the embodiment of the present invention makes it possible to improve the measurement accuracy of the target included in the sample as compared with the conventional case.
- a fluorescence-modified PCR product obtained by performing PCR on a DNA sample using a fluorescence-modifying primer is added to a DNA microarray and hybridized, and the sample is mixed. It detects the target of.
- the method disclosed in Non-Patent Document 2 described above is to detect a target in a sample by hybridizing and reacting a target immobilized on a microarray with a fluorescence-modified fluorescent probe.
- the method disclosed in Patent Document 1 described above is a nucleic acid sequence measurement device provided with a fluorescent probe to which a fluorescent molecule is added and a quenching probe to which a quenching molecule for quenching the fluorescence of the fluorescent molecule is added as a detection probe.
- DNA microarray is used to measure the target. In this method, it is possible to measure the target without adding fluorescent molecules to the target and washing the DNA microarray (washing for removing uncollected targets and the like).
- a target in a nucleic acid sequence measuring device that measures the presence or absence or amount of a specific nucleic acid using a DNA microarray, when a target is not present, donor fluorescent probes and quenching probes that are independent of each other are bound via a binding portion. It is maintained and the fluorescence of the fluorescent molecule is quenched by the quenching molecule.
- the target binds to the detection part, the bond between the donor fluorescent probe and the quenching probe via the binding part is broken, and the quenching molecule separates from the donor fluorescent molecule, so that the donor fluorescent molecule fluoresces. Present.
- the target contained in the sample can be measured.
- the nucleic acid sequence measurement method of Non-Patent Document 1 described above requires a washing operation for removing a target that has not been collected, and the reaction target is exfoliated by the washing operation, resulting in a decrease in the quantitativeness of the target.
- the lower limit of detection may worsen.
- the nucleic acid sequence measurement method of Non-Patent Document 2 requires a washing operation for removing uncollected probes, and the reacted probe is peeled off by the washing operation, the quantitativeness of the target is lowered, and the lower limit of detection is reached. May get worse.
- Non-Patent Document 1 and Non-Patent Document 2 there is a possibility that the sample is mixed between adjacent wells on the microarray due to the washing operation required for the method, and the target cannot be detected accurately. There is.
- Patent Document 1 it is possible to measure a target without cleaning the DNA microarray (cleaning for removing a target or the like that has not been collected).
- nucleic acid samples extracted from a sample containing a living body or a microorganism as a target preparation often contain residual molecules such as proteins and sugars derived from the sample. Therefore, the nucleic acid sample solution emits fluorescence when acquiring a fluorescent image, which causes an increase in the amount of background light, and offset light is emitted from the sample solution supplied to the nucleic acid sequence measurement device. .. Such offset light becomes noise and deteriorates the measurement accuracy.
- the fluorescence emitted from the solution of the sample supplied to the nucleic acid sequence measurement device is also weak, and when this weak fluorescence is buried in the offset light, the target is measured. I can't do it.
- a conjugate of the target and a capture molecule that specifically binds to the target (hereinafter, may be simply referred to as “capture molecule”) modified with a fluorescent molecule is provided.
- a solution containing an excitation light that excites the fluorescent molecule or an absorbent substance that absorbs the fluorescence emitted from the fluorescent molecule (hereinafter, may be simply referred to as “absorbent substance”) is in contact with the solid phase surface of the substrate.
- the fluorescence obtained by irradiating the excitation light that excites the fluorescent molecule from the side opposite to the solid surface surface of the substrate is measured from the side opposite to the solid phase surface of the substrate. ..
- the measurement accuracy of the target included in the sample can be improved as compared with the conventional case.
- a method for obtaining a conjugate of the target and a capture molecule specifically bound to the target which is modified with a fluorescent molecule.
- Examples thereof include a method of supplying the other and the fluorescent molecule to obtain the conjugate.
- a capture molecule modified with the fluorescent molecule and the capture molecule are used as a method for obtaining the conjugate by binding the target to the capture molecule modified with the fluorescent molecule, which is immobilized on the solid phase surface.
- the quenching molecule that specifically binds to the quenching molecule modified with the quenching substance is solidified so that the fluorescent molecule modifying the capturing molecule is extinguished by the quenching substance modifying the quenching molecule. It is immobilized on the phase surface so that when the target does not exist, fluorescence does not occur even if the fluorescent molecule is excited by excitation light, and when the target exists, the target is bound to the capture molecule to form a conjugate. There is a way to get it.
- the quenching substance modifying the quenching molecule separates from the fluorescent molecule modifying the capture molecule, so that fluorescence is emitted from the capture molecule immobilized on the solid phase surface. Occur.
- the conjugate by supplying either the target or the capture molecule and the fluorescent molecule to either the target or the capture molecule immobilized on the solid phase surface.
- a nucleic acid having a specific nucleic acid sequence and a nucleic acid probe having a nucleic acid sequence complementary to the specific nucleic acid sequence of the nucleic acid is immobilized on the solid phase surface of the nucleic acid and the nucleic acid probe.
- Examples thereof include a method of supplying either one and an intercalator modified with a fluorescent molecule to bind to the nucleic acid and the conjugate of the nucleic acid probe to the solid phase surface to obtain a conjugate.
- the target is not particularly limited as long as it is a target to be detected in the sample, and examples thereof include nucleic acids such as DNA and RNA, peptides, and proteins.
- the capture molecule that specifically binds to the target include a detection probe that hybridizes with nucleic acid, an antibody or antibody fragment that specifically binds to an antigen such as a peptide or protein, and an aptamer that specifically binds to nucleic acid.
- the antibody either a polyclonal antibody or a monoclonal antibody can be used, but a monoclonal antibody is preferable.
- the antibody fragment examples include F (ab') 2 , F (ab) 2 , Fab', Fab, Fv, scFv, variants thereof, fusion proteins or fusion peptides containing an antibody moiety, and the like.
- the target may be an antibody or an antibody fragment
- the capture molecule may be an antigen such as a peptide or protein that specifically binds to the antibody or antibody fragment.
- the combination of the target and the capture molecule that specifically binds to the target is, for example, a detection probe in which the target is a nucleic acid having a specific nucleic acid sequence and the capture molecule has a sequence complementary to the specific nucleic acid sequence.
- a detection probe in which the target is a nucleic acid having a specific nucleic acid sequence and the capture molecule has a sequence complementary to the specific nucleic acid sequence.
- the target is an antigen and the capture molecule is an antibody or an antibody fragment that specifically binds to the antigen.
- the target is a nucleic acid having a specific nucleic acid sequence and the capture molecule is a detection probe having a sequence complementary to the specific nucleic acid sequence
- the target nucleic acid is hybridized to the detection probe which is the capture molecule. Join.
- FIG. 1 shows a specific example of a method of modifying the target with the fluorescent molecule and binding the target to the capture molecule immobilized on the solid phase surface to obtain the conjugate.
- target 3 is DNA having a specific nucleic acid sequence
- the DNA is modified with fluorescent molecule 4
- the capture molecule is a nucleic acid sequence complementary to the specific nucleic acid sequence of target 3.
- It is a DNA probe 1 having a detection sequence 2.
- the DNA probe 1 is immobilized on a DNA microarray 5 which is a substrate via a linker 21.
- the target 3 modified with the fluorescent molecule 4 binds to the DNA probe 1 immobilized on the DNA microarray 5 by a hybridization reaction, and the fluorescent molecule 4 modifying the target 3 is excited by excitation light to cause fluorescence. Be radiated.
- FIG. 2 shows a specific example of a method of modifying the capture molecule with the fluorescent molecule and binding the capture molecule to the target immobilized on the solid phase surface to obtain the conjugate.
- the target 3 is a DNA having a specific nucleic acid sequence
- the capture molecule is a DNA probe 1 having a detection sequence 2 which is a nucleic acid sequence complementary to the specific nucleic acid sequence of the target 3.
- the DNA probe 1 is modified with the fluorescent molecule 4, and the target 3 is immobilized on the DNA microarray 5 which is a substrate.
- the DNA probe 1 modified with the fluorescent molecule 4 binds to the target 3 immobilized on the DNA microarray 5 by a hybridization reaction, and fluoresces by exciting the fluorescent molecule 4 modifying the DNA probe 1 with excitation light. Is radiated.
- FIG. 3 shows a specific example of a method of modifying the capture molecule with the fluorescent molecule and binding the target to the capture molecule immobilized on the solid phase surface to obtain the conjugate.
- the detection sequence 2 is a DNA in which the target 3 has a specific nucleic acid sequence and the capture molecule is a nucleic acid sequence complementary to the specific nucleic acid sequence of the target 3 modified with the fluorescent molecule 4.
- It is a donor fluorescent probe 6 having.
- the donor fluorescent probe 6 is immobilized on a DNA microarray 5 which is a substrate via a linker 21, and a quenching probe 7 having a sequence complementary to the nucleic acid sequence of the donor fluorescent probe 6 modified with the quenching substance 8 is provided.
- the DNA microarray 5 which is a substrate so as to hybridize with the donor fluorescent probe 6 at the binding portion 22.
- the fluorescent molecule 4 is in a state of being extinguished by the quenching substance 8, and no fluorescence is generated even if the fluorescent molecule 4 is excited by the excitation light.
- the target 3 binds to the detection sequence 2 of the donor fluorescent probe 6 modified with the fluorescent molecule 4 by a hybrid reaction, and the extinguishing probe 7 separates from the donor fluorescent probe 6 to cause a fluorescent molecule. Fluorescence is emitted by exciting the fluorescent molecule 4 modifying the donor fluorescent probe 6 modified with 4 with excitation light.
- complementary in the present invention means that one nucleic acid sequence has a nucleic acid sequence capable of forming a double-stranded state with the other nucleic acid sequence, and is not necessarily completely complementary. May contain some mismatched base pairs.
- the fluorescent molecule used in the present invention is not particularly limited as long as it is a molecule that is excited by specific excitation light to generate fluorescence, but Alexa Fluor (registered trademark) series, ATTO series, Brilliant series, and Chromeo (registered trademark).
- Alexa Fluor registered trademark
- ATTO ATTO series
- Brilliant series Brilliant series
- Chromeo registered trademark
- Examples include the series, Bactiochlorin series, FAM, TAMRA, Cy dye series, FITC, HiLite Fluor (registered trademark) series, Rhodamine series, Tide Fluor (registered trademark) series, iFluor (registered trademark) series, and DY dye series.
- the substrate used in the present invention plate-shaped quartz, glass, silicon, single crystals such as calcium fluoride and sapphire, ceramics, resin materials and the like having a rectangular shape when viewed in a plan view are used.
- the resin material include COP (cycloolefin polymer), COC (cyclic olefin copolymer), polycarbonate, acrylic resin, polyethylene resin and the like having excellent optical properties, chemical and thermal stability.
- the shape of the substrate when viewed in a plan view may be any shape.
- the fluorescence obtained by irradiating the excitation light that excites the fluorescent molecule from the side opposite to the solid surface of the substrate is measured from the side opposite to the solid surface of the substrate.
- the solid-phase surface of the substrate to which the target and the capture molecule are bonded is held so that the solid-phase surface of the substrate is in contact with the solution containing the absorbent substance.
- the target in the sample solution is modified with a fluorescent molecule and the capture molecule that specifically binds to the target is immobilized on the solid phase surface of the substrate
- the target modified with the fluorescent molecule in the sample solution Bonds to the trapping molecule immobilized on the solid surface of the substrate.
- the solid phase surface of the substrate to which the target modified with the fluorescent molecule and the capture molecule are bonded is held so that the solid phase surface of the substrate is in contact with the solution containing the absorbent substance.
- the capture molecule that specifically binds to the target in the sample solution is modified with a fluorescent molecule, and when the target is immobilized on the solid phase surface of the substrate, the target is modified with the fluorescent molecule in the sample solution.
- the trapping molecule binds to the target immobilized on the solid surface of the substrate.
- the solid-phase surface of the substrate to which the capture molecule modified with the fluorescent molecule and the target are bonded is held so that the solid-phase surface of the substrate is in contact with the solution containing the absorbent substance.
- the solid-phase surface of the substrate is brought into contact with the container in which the solution is held.
- a method of arranging the substrate in a container a method of injecting the solution into a container integrally prepared with the substrate so that the solid phase surface of the substrate is inside the container, and the like so that the solid phase surface of the substrate comes into contact with the solution.
- the container into which the solution is injected.
- the absorbent substance may be added to the container in advance so that the absorbent substance is added to the sample solution when the sample solution containing the target or the capture molecule is injected into the container.
- the absorbent substance may be added to the sample solution containing the trapping molecule and then injected into the container.
- the timing of adding the absorbent substance to the sample solution may be any stage as long as it is before the fluorescent molecule is irradiated with the excitation light.
- the target and the capture molecule are on the solid surface of the substrate. It may be a stage before the bond on the surface, or it may be a stage after the target and the trapping molecule are bonded on the solid surface surface of the substrate.
- the substrate or the capture molecule is modified with a fluorescent molecule, and the solid phase surface of the substrate to which the target and the capture molecule are bonded is placed in a solution containing an absorbent substance that absorbs the excitation light that excites the fluorescent molecule.
- a solution containing an absorbent substance that absorbs the excitation light that excites the fluorescent molecule By holding the solid phase surface of the solution in contact with each other, when the solution is irradiated with the excitation light that excites the fluorescent molecule, the absorbent substance contained in the solution absorbs the excitation light that tries to pass through the solution. , Transmission of excitation light can be suppressed. By suppressing the transmission of the excitation light into the solution, the excitation of the fluorescent molecules free in the solution can be suppressed, the generation of fluorescence from the solution can be suppressed, and the background light can be reduced.
- the light-absorbing substance When a substance that absorbs fluorescence emitted from a fluorescent molecule is used as the light-absorbing substance, even if the fluorescent substance in the solution is excited by excitation light to emit fluorescence, the light-absorbing substance is released from the solution. By absorbing the emitted fluorescence, it is possible to suppress the generation of fluorescence from the solution and reduce the background light.
- residual molecules such as proteins and sugars derived from the sample contained in the sample solution extracted from the sample containing a living body or a microorganism may be contained in the solution containing the target.
- the light-absorbing substance can suppress the generation of excitation or fluorescence of the residual molecule, it is generated from the residual substance even when applied to a target detection device that originally does not require cleaning.
- the background light due to fluorescence can be reduced, and the detection sensitivity can be further improved.
- the absorbent material is not particularly limited as long as it has an excitation light that excites a fluorescent molecule or an optical property that absorbs light having a wavelength of fluorescence emitted from the fluorescent molecule, and the fluorescent molecule used in the present invention is generated. Although it is appropriately selected according to the wavelength of fluorescence, pigments and the like used for coloring paints, inks, cosmetics, foods and the like are used. Pigments include various particles such as metals such as gold and silver, oxides such as iron oxide, nitrides, and organic polymers. Absorption is selected from the absorption characteristics of the material itself, colored particles, and the optical characteristics of absorption due to surface coloring. It can be used as a substance. In addition, metal nanoparticles can be used as an absorbent because surface plasmon resonance occurs at a specific wavelength due to the interaction between electrons and light on the particle surface and strong dimming of light occurs.
- the absorbent substance used in the present invention include, for example, when Cy3 (registered trademark) is used as a fluorescent molecule, iron oxide (Fe 2 O 3 , Fe 2 O 4 ), gold nanoparticles, silver nanoparticles, and the like.
- Examples thereof include black colored silica particles, polymer black colored particles such as a styrene and acrylic acid copolymer, and the like.
- the absorbent material may be in a dry state or in a solution state.
- the absorbent substance is a substance that absorbs the excitation light
- an absorbent substance that penetrates the solid phase and reduces the scattering of the excitation light that enters the solution containing the absorbent substance is preferable. This is because if the scattering of the excitation light entering the solution containing the absorbent substance is small, the light path length is extended due to the scattering of the light transmitted through the solution due to the scattering substance, and the fluorescent molecules in the solution are added. This is because it is possible to further suppress the phenomenon of being excited by the substance and increasing the fluorescence.
- This phenomenon expresses the logarithm of the ratio of the incident light and the transmitted light transmitted through the substance as the absorbance, and has the effect of scattering on the Lambert-Beer law, which is a law that formulates a proportional relationship with the concentration and the optical path length. Similar to the phenomenon formulated by the added Modified Lambert-Beer law, a phenomenon in which when a scattering substance is present, the increase in absorbance in proportion to the concentration of the substance and the linear optical path length in the optical axis direction cannot be obtained. Is. The scattering phenomenon of light by a substance differs depending on the particle size.
- the absorbent substance For a substance sufficiently larger than the wavelength, it is a geometrical optical approximation, for a particle size of about the wavelength, Mie scattering, and for a particle size sufficiently smaller than the wavelength, Rayleigh scattering. expressed.
- Mie scattering since the absorbent substance is added and dispersed in the solution, it is considered that Mie scattering or Rayleigh scattering occurs. It is known that the total scattering intensity of Mie scattering varies depending on the particle size and increases in proportion to the square to the sixth power of the particle size. It is also known that the total scattering intensity of Rayleigh scattering increases in proportion to the sixth power of the particle size. For the above reasons, in the present invention, it is preferable that the particle size of the absorbent substance is small.
- the absorbent substance is a substance that absorbs fluorescence
- the fluorescence from the fluorescent molecule near the contact surface between the solution and the solid phase surface is faster than the fluorescence from the fluorescent molecule inside the solution
- the solid phase surface of the substrate is Those in which the fluorescence generated near the contact surface between the solution and the solid phase is scattered on the solution side and is absorbed by the absorbent substance without reaching the detector side in order to reach the detector installed on the opposite side. Therefore, an absorbent substance that reduces the scattering of fluorescence is preferable.
- the absorbent substance is added to the solution, it is desirable that it is stably dispersed during the measurement of fluorescence and that unevenness does not occur in the solution.
- the dispersion stability of particles is measured by various measuring methods such as visual observation, measurement of change in transmitted light intensity, and measurement of change in scattered light intensity.
- the difficulty of precipitation can be estimated from the parameters of the centrifugation conditions for centrifuging the substance by a centrifuge, and the dispersion stability can be estimated.
- the particle size is less than 800 nm
- the parameter required for centrifugation is 10,000 ⁇ g, which is 20 minutes, and it is difficult to precipitate, and it is excellent in dispersibility, which is preferable.
- the particle size is 200 nm or less, when it is iron oxide particles, the particle size is 100 nm or less, and when it is gold nanoparticles, particles having a particle size of 15 nm or less are excellent in dispersibility and are preferable.
- the absorbent substance is preferably hydrophilic in order to be added to and dispersed in the solution.
- the absorptive substance is hydrophobic, it can be modified to be hydrophilic on the surface, introduced with surface functional groups such as carboxyl groups and sulfon groups, coated with oxides, and hydrophilic polymers such as PEG, PEO and dextran. It is preferable to make it hydrophilic by chemical modification or the like.
- the added absorbance substance does not adsorb the target or the capture molecule.
- the hydrophilic polymer such as PEG
- a blocking agent such as BSA that suppresses non-specific adsorption of biomolecules to a solution containing an absorbent substance, it is possible to suppress non-specific adsorption of the absorbent substance on a target or a trapping molecule.
- the excitation light that excites the fluorescent molecule is irradiated from the side opposite to the solid surface surface of the substrate, and the obtained fluorescence is measured from the side opposite to the solid surface surface of the substrate.
- the method of irradiating the excitation light that excites the fluorescent molecule from the side opposite to the solid phase surface of the substrate and measuring the obtained fluorescence is not particularly limited as long as the fluorescence emitted from the fluorescent molecule can be measured.
- a method of measuring by using a camera and binding a fluorescent image generated from a fluorescent molecule onto a detection element of the camera can be mentioned.
- the excitation light source examples include a laser light source that emits a single-wavelength laser beam or its expanded light, an LED (Light Emitting Diode), a lamp that emits white light, a light source composed of a combination of an LED and a wavelength filter, and the like. Can be used.
- a laser light source that emits a single-wavelength laser beam or its expanded light
- an LED Light Emitting Diode
- a lamp that emits white light a light source composed of a combination of an LED and a wavelength filter, and the like.
- the camera used for measurement can be a color or monochrome CCD, a CMOS camera, an EM-CCD characterized by high sensitivity, a digital CMOS, or the like. Further, a combination such as a photodiode which is a single detector arranged one-to-one with the spot may be used.
- the fluorescent image obtained by the target measurement method of the present invention it is possible to acquire images before and after the binding between the target at the same spot and the captured molecule. Therefore, it is not affected by variations in the amount of light between solid phases and spots.
- the amount of change in fluorescence can be calculated from the fluorescence images before and after binding, and the number of bound molecules can be calculated.
- the calculation of the fluorescence change amount may use the average light amount of the entire spot, or may use the fluorescence change amount of each pixel of the spot image.
- the target measurement device of the present invention can be used in the target measurement method of the present invention.
- the target measurement device of the present invention is a target measurement device used when measuring a target contained in a sample, and is a substrate in which either the target or the capture molecule is immobilized on a solid phase surface. And the container to which the excitation light that excites the fluorescent molecule that modifies the bond between the target and the capture molecule, or the absorbent substance that absorbs the fluorescence emitted from the fluorescent molecule is added, and the target and the capture.
- a device for target measurement comprising one of the molecules and a container capable of holding a solution containing the absorbent substance in contact with the solid phase surface of the substrate.
- the target measurement device of the present invention is a target measurement device in which a trapping molecule is immobilized on a solid phase surface of a substrate and a solution containing a target modified with a fluorescent molecule is supplied to a container; Target measurement device in which a target is immobilized on a solid surface and a solution containing a capture molecule modified with a fluorescent molecule is supplied to a container; a capture molecule modified with a fluorescent molecule on the solid surface of a substrate. Is immobilized and a target measuring device is supplied with a solution containing the target in a container; one of a target and a capture molecule is immobilized on the solid phase surface of the substrate, and the container is equipped with the above.
- Examples thereof include a target measurement device to which a solution containing any one of the target and the capture molecule and a fluorescent molecule that binds to a conjugate of the target and the capture molecule is supplied.
- Examples of the target and the capture molecule include those described above.
- FIG. 4 is a diagram showing an example of the configuration of the target measurement device of the present invention.
- FIG. 4 shows an example of a DNA microarray on which a DNA probe is immobilized as a solid phase.
- a target or a trapping molecule is immobilized on the solid phase surface of the substrate.
- a DNA probe 1 having a complementary sequence of a specific target nucleic acid sequence is immobilized on a DNA microarray 5 which is a substrate.
- the target measurement device of the present embodiment is emitted from the target 3 in which the target 3 is modified with the fluorescent molecule 4 and the fluorescent molecule 4 is modified, and the excitation light 33 for exciting the fluorescent molecule 4 or the fluorescent molecule 4.
- a container 32 capable of holding an absorbent target solution 35 containing an absorbent substance that absorbs fluorescence 34, and a DNA microarray 5 in which the absorbent substance-added target solution 35 is immobilized with the DNA probe 1. It is held in contact with the solid phase surface of.
- FIG. 5 is a flowchart showing an operation procedure for detecting the target 3 using the target measurement device shown in FIG.
- the DNA probe 1 modified with the fluorescent molecule 4 is immobilized on the DNA spot 30 (step S1).
- the target 3 in the sample is modified with the fluorescent molecule 4 to prepare a target solution (step S2).
- amplification of the target 3 having a specific nucleic acid sequence may be performed.
- the timing for confirming whether or not the target 3 is present in the sample is not limited to that after the completion of amplification, and may be during amplification.
- the modification of the target 3 with the fluorescent molecule 4 may be performed after the amplification is confirmed, and the process may proceed to step S3 described later only when the amplification is confirmed.
- electrophoresis, antigen-antibody reaction, mass spectrometry, real-time PCR and the like can be appropriately used.
- the prepared target solution is supplied to the container 32 to which the DNA microarray on which the DNA probe 1 is immobilized is arranged in the container to which the light-absorbing substance is added, and the target solution is supplied to the DNA. It is brought into contact with the solid surface of the microarray 5 (step S3).
- the absorbent substance added to the container 32 is added to the target solution when the target solution is supplied to the container 32, and becomes the absorbent substance-added target solution 35.
- the target 3 modified with the fluorescent molecule 4 and the DNA immobilized on the DNA microarray 5 Hybridization reaction with probe 1 (step S4).
- the target 3 binds to the DNA probe 1, and the fluorescent molecule 4 modifying the target 3 is captured in the DNA spot 30 on which the DNA probe 1 is immobilized.
- the excitation light 33 that excites the fluorescent molecule 4 is irradiated from the side opposite to the solid phase surface of the DNA microarray 5 (step S5).
- the fluorescence 34 generated from the fluorescent molecule 4 that modifies the target 3 bound to the DNA probe 1 immobilized on the DNA microarray 5 is detected from the side opposite to the solid phase surface of the DNA microarray 5 (step S6). ..
- the fluorescence image generated from the fluorescence molecule 4 is acquired by the fluorescence reader 40.
- the amount of fluorescence is calculated from the acquired fluorescence image (step S7).
- the light-absorbing substance is a substance that absorbs the excitation light 33 that excites the fluorescent molecule 4
- the excitation light 33 for exciting the fluorescent molecule 4 is irradiated to the light-absorbing substance-added target solution 35, but the light-absorbing substance-added target Since the solution 35 contains an absorbent substance that absorbs the excitation light 33 that excites the fluorescent molecule 4, the unreacted fluorescent molecule 4 that is free in the solution 35 and does not hybridize with the DNA probe 1 is released. It is possible to suppress the excitation of the fluorescent molecule 4 of the target 3 modified with.
- the excitation light 33 for exciting the fluorescent molecule 4 is irradiated to the target solution 35 to which the light-absorbing substance is added, but the light-absorbing substance is added. Since the target solution 35 contains an absorbent substance that absorbs the fluorescence 34 emitted from the fluorescent molecule 4, the unreacted fluorescent molecule 4 that is free in the solution 35 and does not hybridize with the DNA probe 1 It is possible to suppress the generation of fluorescence from the fluorescent molecule 4 of the target 3 modified with.
- the number of molecules of the hybridized target 3 can be calculated from the amount of change in fluorescence of the fluorescent molecule 4 before and after the hybridization reaction.
- a hybrid reaction is carried out using a standard solution of Target 3 having a known number of molecules, the amount of change in fluorescence of fluorescent molecule 4 before and after the reaction is measured, and a calibration curve showing the relationship between the number of molecules and the amount of change in fluorescence. Is created in advance. From this calibration curve and the amount of change in fluorescence of the fluorescent molecule 4 before and after the hybridization reaction using the sample, the number of molecules of the hybridized target 3 can be calculated.
- an absorbent substance is added to the solution in step S2 or step S3.
- a solution containing a target or a capture molecule that specifically binds to the target is prepared, and the concentration of the target or the capture molecule is adjusted.
- the concentration of the target or the capture molecule is adjusted. For example, if the target is DNA having a specific nucleic acid sequence and the capture molecule is a DNA probe having a complementary sequence to the specific nucleic acid sequence of the target, a DNA probe solution is prepared and the DNA probe solution is prepared. Adjust the concentration.
- the target or trapping molecule is immobilized on the solid surface of the substrate.
- the DNA probe is immobilized on the solid surface
- the DNA probe 1 is spotted on the solid surface using a spotter or the like, and the DNA probe 1 is immobilized on the solid surface.
- the solid phase surface is immersed in a blocking solution to inactivate unreacted active functional groups.
- the region on the solid phase surface where the target or the trapping molecule is spotted may be divided into blocks in units of a predetermined number.
- the addition of the target solution to the target measurement device is performed block by block. Further, the image acquisition of the target measurement device is often performed for each block. That is, it can be said that the block is an image acquisition area.
- the solid phase surface is washed to remove excess unimmobilized targets or trapping molecules, and the washing liquid is also removed.
- the substrate produced by the above procedure is appropriately stored until use in an environment suitable for the substrate and the properties of the target and the trapping molecule immobilized on the substrate, such as shading, temperature, and humidity conditions.
- the substrate on which the target or capture molecule is immobilized in (3) has a solid phase surface on which the target or capture molecule is immobilized.
- the substrate may be integrated with the container, or the substrate and the container may be separated so that the substrate can be arranged in the container when measuring the target.
- the absorbent material may be in a dry state or in a liquid state.
- the absorbent substance can be added to the solution when the solution containing the target or the capture molecule modified with the fluorescent molecule is supplied to the container.
- the absorption substance may be added to the container before the substrate is placed in the container, or may be added after the substrate is placed in the container. When the absorbent substance is added after the substrate is placed in the container, the absorbent substance can be added from the injection port provided in the container.
- the target measuring device of the present invention includes a target measuring device of the present invention and a fluorescence reading device for measuring the amount of fluorescence of a fluorescent molecule from the target measuring device.
- FIG. 6 is an example of a configuration diagram showing the target measuring device of the present invention.
- the target measuring device 10 since the target of the target measuring device 10 and the image before and after the binding of the captured molecule are acquired, after the image before the binding is acquired, the target measuring device 10 is fixed by the temperature control stage. The temperature of the phase surface is raised to allow the bonding reaction to proceed, and the image after bonding is acquired in a state where the temperature is lowered to room temperature again.
- the target 3 modified with the fluorescent molecule 4 is hybridized with the DNA probe 1 to obtain images before and after the hybridization reaction.
- the temperature control stage has a shaking function, rotation of a target measuring device, stirring function by a vortex mixer, etc. during the reaction between the target and the trapped molecule in order to promote the bond between the target and the trapped molecule.
- the laser light emitted from the laser light source 41 is reflected by the dichroic mirror 44 via the mirror 45 and illuminates the solid phase surface of the target measurement device.
- the irradiated light becomes the excitation light 33 for the fluorescent molecule 4 on the solid phase surface of the target measurement device 10, the fluorescent molecule 4 is in an excited state, and the fluorescent molecule 4 radiates the fluorescence 34.
- the fluorescence emitted from the solid phase surface of the target measurement device 10 passes through the dichroic mirror 44 and is detected by forming a fluorescence image on the detection element of the CCD camera 42 via the imaging optical system 43.
- a bandpass filter tuned to the excitation light wavelength may be installed on the excitation light 33 side, or tuned to the fluorescence wavelength to be detected on the fluorescence 34 side.
- a band pass filter may be installed.
- the fluorescent image obtained by the target measuring device of the present invention can acquire images before and after the binding between the target at the same spot and the captured molecule. Therefore, it is not affected by variations in the amount of light between solid phases and spots.
- the amount of change in fluorescence can be calculated from the fluorescence images before and after the binding reaction, and the number of molecules undergoing the binding reaction can be calculated.
- the calculation of the fluorescence change amount may use the average light amount of the entire spot, or may use the fluorescence change amount of each pixel of the spot image.
- the target measuring device of the present invention may include a computer for controlling the CCD camera 42, an arithmetic device for calculating the light amount of the image, and a recording device for storing the image and the light amount.
- the target measuring device of the present invention is not limited to the above embodiment. Since the target measuring device of the present invention detects fluorescence from the surface opposite to the fixed surface of the detection spot on the solid phase surface, a fluorescence microscope, a confocal microscope, an evanescent fluorescence detector, a thin film oblique illumination microscope, and a sheet illumination microscope. , A structured illumination microscope, a multiphoton excitation microscope, etc. can be used.
- the target measurement kit of the present invention can be used for the target measurement method of the present invention.
- the target measurement kit of the present invention is a target measurement kit used when measuring a target contained in a sample, and is a substrate in which either the target or the capture molecule is immobilized on a solid phase surface. And a container that can hold a solution containing either the target or the capture molecule in contact with the solid phase surface of the substrate, and fluorescence that modifies the conjugate of the target and the capture molecule. It contains an excitation light that excites a molecule or an light-absorbing substance that absorbs the fluorescence emitted from the fluorescent molecule.
- the absorbent material may be added to the sample solution when preparing the sample solution containing the target or capture molecule.
- the timing of adding the absorbent substance to the sample solution may be any stage as long as it is before the fluorescent molecule is irradiated with the excitation light. For example, even at the stage of preparing the sample solution, the target and the capture molecule are bonded on the solid phase. It may be a previous step, or it may be a step after the target and the capture molecule are bound on the solid phase.
- An absorbent substance may be added to the container in advance.
- the absorbent material may be in a dry state or in a liquid state.
- the absorbent substance can be added to the solution when the solution containing the target or the capture molecule modified with the fluorescent molecule is supplied to the container.
- a capture molecule is immobilized on the solid phase surface of the substrate, and the container is in a state where a solution containing the target modified with fluorescent molecules and the light-absorbing substance is brought into contact with the surface of the substrate.
- the target may be immobilized on the solid phase surface of the substrate, and the container may contain a capture molecule modified with a fluorescent molecule and the light-absorbing substance. It may be a container capable of holding the containing solution in contact with the solid phase surface.
- a capture molecule modified with a fluorescent molecule is immobilized on the solid phase surface of the substrate, and the container is brought into contact with the solid phase surface by a solution containing the target and the absorbent substance. It may be a container that can be held in a state.
- either one of the target and the trapping molecule is immobilized on the solid surface of the substrate, and the container has the target and the trapping molecule on the other side of the target and the trapping molecule.
- a container may be used in which a solution containing the fluorescent molecule bound to the conjugate of the substrate and the light-absorbing substance can be held in contact with the solid phase surface of the substrate.
- Examples of the target and capture molecule include the above-mentioned target and capture molecule.
- Examples of the target measurement kit of the present invention include a kit having a specific nucleic acid sequence as a target and a DNA probe having a sequence complementary to the specific nucleic acid sequence as a capture molecule. ..
- the target measurement kit of the present invention may include the target measurement device of the present invention.
- the above-mentioned substrates, absorbent substances, and containers can be mentioned.
- the target measurement kit of the present invention may further include a standard solution necessary for quantifying the target, a necessary buffer solution, a product description, and the like.
- the target is DNA having a specific nucleic acid sequence
- the capture molecule is a DNA probe having a sequence complementary to the specific nucleic acid sequence.
- the DNA probe is immobilized on the solid surface of a DNA microarray, and the target is modified with a fluorescent molecule.
- the target measurement kit of the present embodiment is a container capable of holding a DNA microarray 5 on which a DNA probe 1 is immobilized and a target solution 35 to which an absorbent substance is added, and the solution 35 is used as the DNA probe 1.
- a container 32 that is held in contact with the solid phase surface of the DNA microarray 5 on which the DNA microarray 5 is immobilized, and an absorbent substance is added to the container 32.
- the target 3 in the sample is modified with the fluorescent molecule 4 to prepare a target solution.
- amplification of the target 3 having a specific nucleic acid sequence may be performed.
- the target solution is supplied to the container 32, and the target solution is brought into contact with the solid phase surface of the DNA microarray 5.
- the absorbent substance added to the container 32 is added to the target solution when the target solution is supplied to the container 32, and becomes the absorbent substance-added target solution 35.
- the target 3 modified with the fluorescent molecule 4 and the DNA immobilized on the DNA microarray 5
- the probe 1 and the probe 1 are hybridized.
- the target 3 binds to the DNA probe 1, and the fluorescent molecule 4 modifying the target 3 is captured in the DNA spot 30 on which the DNA probe 1 is immobilized.
- the excitation light 33 that excites the fluorescent molecule 4 is irradiated from the side opposite to the solid phase surface of the DNA microarray 5.
- the fluorescence 34 generated from the fluorescent molecule 4 that modifies the target 3 bound to the DNA probe 1 immobilized on the DNA microarray 5 is detected from the side opposite to the solid phase surface of the DNA microarray 5.
- the fluorescence image generated from the fluorescence molecule 4 is acquired by the fluorescence reader 40. Then, the amount of fluorescence is calculated from the acquired fluorescence image.
- the scope of application of the present invention is not limited to the above embodiment.
- the present invention can be widely applied to a target measurement method for measuring a target contained in a sample, a target measurement device, a target measurement device, and a target measurement kit.
- the target measurement method, target measurement device, target measurement device, and target measurement kit of the present invention include dry image measurement in fluorescent molecular light intensity measurement, in-liquid observation of fluorescent molecular light intensity of biochip, and real-time observation in continuous reaction. Can be used for. Specifically, it can be used, for example, for bacterial species discrimination by gene / polymer analysis, oncogene, animal and plant discrimination, intestinal bacterial test, and the like.
- the target measurement method, the target measurement device, the target measurement device, and the target measurement kit of the present invention are also applied to a solid phase method such as a labeled antibody method used for clinical examinations and the like.
- a solid phase method such as a labeled antibody method used for clinical examinations and the like.
- the FISH method fluorescence in situ hybridization
- the FIA method fluorescence immunofluorescence measurement method
- the IFA measures the serum (antibody) reaction that labels the fluorescent substance on the pathogen that becomes the antigen. It also applies to the method (indirect fluorescent antibody method).
- Example 1 The effect of reducing the background light by adding the absorbent substance was confirmed. Cy3® molecules were adjusted to a concentration of 0.3 to 3,000 nM as fluorescent molecules 4 in the container, and a transparent glass substrate was placed on the container. A fluorescent image was acquired by transmitting the excitation light 33 at 532 nm through the transparent glass substrate.
- FIG. 7 shows the results of calculating the amount of background light from the acquired fluorescent image when the absorbent substance was added to the solution in the container and when it was not added.
- Table 1 shows the background light ratio (the amount of background light when the absorbent substance is not added / the amount of background light when the absorbent substance is added) when the concentration of the fluorescent molecule is changed.
- the light-absorbing substance used was dextran-coated iron oxide (Fe 2 O 3 ) having a particle size of 50 nm, and was added so as to be 50 mg / ml in the solution.
- the background light can be reduced from 1/4 to 1/17 depending on the Cy3 molecular concentration, and the reduction effect is high when the background light is high.
- the fluorescence of the solution is about 10 ⁇ W / m 2 , and the effect is obtained even in the solution with the background light of about 10 ⁇ W / m 2 , from the sample. It was found that the fluorescence generated by exciting the solution generated by molecules other than the brought-in target 3 is also effective.
- the background light can be reduced to 1/2 even in the state of water containing no Cy3 molecule, but this is because the excitation light 33 does not pass through the solution, so that the reflection on the bottom surface of the container and the autofluorescence are reduced. It is considered that this is the cause, and a secondary effect was confirmed.
- FIG. 8 is a spot image of a spot in which a synthetic DNA modified with Cy3 molecules is immobilized on a substrate and has an exposure time of 1 second by a fluorescence reader in a solution having a Cy3 molecule concentration of 3 to 300 nM. It was found that when the Cy3 molecular concentration is 30 nM or more, the background light can be reduced to 1/10 or more, so that spot observation is possible even when the fluorescence exhibited by the solution is high.
- FIG. 9 shows the relationship between the amount of spot light and the background light when the Cy3 molecular concentration is 30 nM.
- the amount of fluorescence due to the fluorescent molecule 4 captured by the DNA probe 1 of the spot is calculated from the difference between the amount of light of the spot and the background light. No, the detection signal was not lowered by adding the absorbent substance.
- the ratio of the amount of spot light to the background light is S / N
- the S / N is 1.3 when the absorbent substance is not added, whereas the S / N is when the absorbent substance is added. It became 5.3, and the S / N was improved by 4.2 times.
- Example 2 A DNA microarray 5 in which a plurality of unmodified DNA probes 1 were arranged on a substrate was prepared, and the applicability of the addition of an absorbent substance was confirmed in spot observation by hybridization of a target DNA modified with a Cy3 molecule as follows. did. A target DNA modified with a Cy3 molecule so as to be 0.25 nM was prepared in a container, and at this time, an absorbent substance was added under the same conditions as in Example 1, and a DNA microarray 5 was placed. Incubation was carried out at 60 ° C. and 5 rpm for 30 minutes to hybridize the DNA probe 1 and the target DNA. After returning to room temperature, a fluorescence image of the spot was acquired by a fluorescence reader, and the amount of light was calculated. The results are shown in FIG.
- the absorbent substance when the absorbent substance was added, the fluorescence of the target modified with the unreacted Cy3 molecule released in the solution was reduced, and the background light was reduced to 1 / 3.7.
- the ratio of the amount of spot light to the background light is S / N
- the S / N when the absorbent substance is not added is 1.5
- the S / N when the absorbent substance is added is 1.5.
- the S / N was improved 2.5 times.
- the absorbent substance is the hybrid reaction between the DNA probe 1 and the target 3. It can be seen that it does not inhibit.
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Abstract
A target measurement method for measuring a target contained in a sample, wherein when excitation light that excites fluorescent molecules, or a solution containing a light-absorbing substance that absorbs the fluorescence emitted by fluorescent molecules contacts a solid-phase surface of a substrate that is provided with a fluorescent molecule-modified bound product of the target and capture molecules that specifically bind to the target, the fluorescence obtained by the irradiation of the excitation light from the opposite side to the solid-phase surface of the substrate is measured from the opposite side to the solid-phase surface of the substrate.
Description
本発明は、ターゲット計測方法、ターゲット計測用デバイス、ターゲット計測装置、及びターゲット計測用キットに関する。
The present invention relates to a target measurement method, a target measurement device, a target measurement device, and a target measurement kit.
サンプルに含まれる特定の核酸配列を有するターゲットを計測する方法として、ＤＮＡマイクロアレイ（特定の核酸配列の相補配列を有する検出プローブが基板等の固相面に設けられたもの）を用いる方法が広く知られている。この方法は、ＤＮＡマイクロアレイに添加されたサンプルに含まれるターゲットが、ハイブリダイズ反応によりＤＮＡマイクロアレイの検出プローブに捕集される性質を利用してターゲットを計測する方法である。この方法では、ターゲットがサンプルに含まれるか否かに加えて、サンプルに含まれるターゲットの量を計測することができる。以下の非特許文献１、２及び特許文献１には、ＤＮＡマイクロアレイを用いてターゲットを計測する従来の計測方法が開示されている。
As a method for measuring a target having a specific nucleic acid sequence contained in a sample, a method using a DNA microarray (a detection probe having a complementary sequence of a specific nucleic acid sequence provided on a solid surface such as a substrate) is widely known. Has been done. This method is a method of measuring a target by utilizing the property that the target contained in the sample added to the DNA microarray is collected by the detection probe of the DNA microarray by the hybridization reaction. In this method, it is possible to measure the amount of the target contained in the sample in addition to whether or not the target is contained in the sample. The following Non-Patent Documents 1 and 2 and Patent Document 1 disclose conventional measurement methods for measuring a target using a DNA microarray.
ところで、上記非特許文献１、２の核酸配列計測方法は、捕集されていないターゲットを除去するための洗浄操作が必要であり、この洗浄操作を行うことによって計測精度が悪化する可能性があるという問題がある。また、上記特許文献１の核酸配列計測方法は、上記非特許文献１、２のような洗浄操作は必要としないものの、ＤＮＡマイクロアレイに供給するサンプルの溶液から発せられるオフセット光がノイズとなって計測精度を悪化させてしまうという問題がある。
By the way, the nucleic acid sequence measuring methods of Non-Patent Documents 1 and 2 require a washing operation for removing a target that has not been collected, and the washing operation may deteriorate the measurement accuracy. There is a problem. Further, although the nucleic acid sequence measurement method of Patent Document 1 does not require the washing operation as in Non-Patent Documents 1 and 2, the offset light emitted from the solution of the sample supplied to the DNA microarray becomes noise and is measured. There is a problem that the accuracy is deteriorated.
本発明は、上記事情に鑑みてなされたものであり、サンプルに含まれるターゲットの計測精度を従来よりも向上させることができるターゲット計測方法、ターゲット計測用デバイス、ターゲット計測装置、及びターゲット計測用キットを提供することを目的とする。
The present invention has been made in view of the above circumstances, and is a target measurement method, a target measurement device, a target measurement device, and a target measurement kit that can improve the measurement accuracy of the target contained in the sample. The purpose is to provide.
上記の目的を達成するために、本発明は以下の構成を採用した。
［１］ サンプルに含まれるターゲットを計測するターゲット計測方法であって、
蛍光分子で修飾された、前記ターゲットと前記ターゲットに特異的に結合する捕捉分子との結合物が設けられた基板の固相面に、前記蛍光分子を励起する励起光、又は前記蛍光分子から発せられる蛍光を吸収する吸光物質を含む溶液が接触している状態で、前記基板の前記固相面とは反対側から前記励起光を照射して得られる蛍光を、前記基板の前記固相面とは反対側から計測するターゲット計測方法。
［２］ 前記ターゲットを前記蛍光分子で修飾し、
前記固相面に固定化されている前記捕捉分子に前記ターゲットを結合させて前記結合物を得る、［１］に記載のターゲット計測方法。
［３］ 前記捕捉分子を前記蛍光分子で修飾し、
前記固相面に固定化されている前記ターゲットに前記捕捉分子を結合させて前記結合物を得る、［１］に記載のターゲット計測方法。
［４］ 前記捕捉分子を前記蛍光分子で修飾し、
前記固相面に固定化されている前記捕捉分子に、前記ターゲットを結合させて前記結合物を得る、［１］に記載のターゲット計測方法。
［５］ 前記固相面に固定化されている、前記ターゲット及び前記捕捉分子のいずれか一方に対し、前記ターゲット及び前記捕捉分子のいずれか他方と前記蛍光分子とを供給して前記結合物を得る、［１］に記載のターゲット計測方法。
［６］ 前記ターゲットは、特定の核酸配列を有する核酸であり、
前記捕捉分子は、前記特定の核酸配列と相補的な配列を有する検出プローブであり、
前記ターゲットを前記検出プローブにハイブリダイズ反応させて前記結合物を得る、［１］～[５]のいずれか１項に記載のターゲット計測方法。
［７］ サンプルに含まれるターゲットを計測する際に用いられるターゲット計測用デバイスであって、
前記ターゲットと前記ターゲットに特異的に結合する捕捉分子とのいずれか一方が固相面に固定化される基板と、
前記ターゲットと前記捕捉分子との結合物を修飾する蛍光分子を励起する励起光、又は前記蛍光分子から発せられる蛍光を吸収する吸光物質が添加された容器であって、前記ターゲット及び前記捕捉分子のいずれか他方と、前記吸光物質とを含む溶液を、前記基板の前記固相面に接触させた状態で保持可能な容器と、
を備えたターゲット計測用デバイス。
［８］ 前記固相面には、前記捕捉分子が固定化されており、
前記容器には、前記蛍光分子で修飾された前記ターゲットを含む溶液が供給される、［７］に記載のターゲット計測用デバイス。
［９］ 前記固相面には、前記ターゲットが固定化されており、
前記容器には、前記蛍光分子で修飾された前記捕捉分子を含む溶液が供給される、［７］に記載のターゲット計測用デバイス。
［１０］ 前記固相面には、前記蛍光分子で修飾されている、前記捕捉分子が固定化されており、
前記容器には、前記ターゲットを含む溶液が供給される、［７］に記載のターゲット計測用デバイス。
［１１］ 前記固相面には、前記ターゲット及び前記捕捉分子のいずれか一方が固定化されており、
前記容器には、前記ターゲット及び前記捕捉分子のいずれか他方と、前記ターゲットと前記捕捉分子との結合体に結合する前記蛍光分子と、を含む溶液が供給される、［７］に記載のターゲット計測用デバイス。
［１２］ 前記ターゲットは、特定の核酸配列を有するターゲットであり、
前記捕捉分子は、前記特定の核酸配列に相補的な配列を有する検出プローブである、［７］～［１１］のいずれか１項に記載のターゲット計測用デバイス。
［１３］ ［７］～［１２］のいずれか１項に記載のターゲット計測用デバイスと、
前記ターゲット計測用デバイスからの蛍光量を測定する蛍光読取装置と、
を有する、ターゲット計測装置。
［１４］ サンプルに含まれるターゲットを計測する際に用いられるターゲット計測用キットであって、
前記ターゲットと前記ターゲットに特異的に結合する捕捉分子とのいずれか一方が固相面に固定化された基板と、
前記ターゲット及び前記捕捉分子のいずれか他方を含む溶液を、前記基板の前記固相面に接触させた状態で保持可能な容器と、
前記ターゲットと前記捕捉分子との結合物を修飾する蛍光分子を励起する励起光、又は前記蛍光分子から発せられる蛍光を吸収する吸光物質と、
を含むターゲット計測用キット。
［１５］ 前記吸光物質は、前記容器に予め添加されている、［１４］に記載のターゲット計測用キット。
［１６］ 前記固相面には、前記捕捉分子が固定化されており、
前記容器には、前記蛍光分子で修飾された前記ターゲットと、前記吸光物質とを含む溶液が保持される、［１４］又は［１５］に記載のターゲット計測用キット。
［１７］ 前記固相面には、前記ターゲットが固定化されており、
前記容器には、前記蛍光分子で修飾された前記捕捉分子と、前記吸光物質とを含む溶液が保持される、［１４］又は［１５］に記載のターゲット計測用キット。
［１８］ 前記固相面には、前記蛍光分子で修飾されている前記捕捉分子が固定化されており、
前記容器には、前記ターゲットと前記吸光物質とを含む溶液が保持される、［１４］又は［１５］に記載のターゲット計測用キット。
［１９］ 前記固相面には、前記ターゲット及び前記捕捉分子のいずれか一方が固定化されており、
前記容器には、前記ターゲット及び前記捕捉分子のいずれか他方と、前記ターゲットと前記捕捉分子との結合体に結合する前記蛍光分子と、前記吸光物質と、を含む溶液が保持される、［１４］又は［１５］に記載のターゲット計測用キット。
［２０］ 前記ターゲットは、特定の核酸配列を有するターゲットであり、
前記捕捉分子は、前記特定の核酸配列に相補的な配列を有する検出プローブである、［１４］～［１９］のいずれか１項に記載のターゲット計測用キット。 In order to achieve the above object, the present invention has adopted the following configuration.
[1] A target measurement method for measuring a target included in a sample.
The excitation light that excites the fluorescent molecule or the fluorescent molecule emits light on the solid phase surface of the substrate provided with the conjugate of the target and the capture molecule that specifically binds to the target, which is modified with the fluorescent molecule. The fluorescence obtained by irradiating the excitation light from the side opposite to the solid phase surface of the substrate in a state where the solution containing the absorbent substance that absorbs the fluorescence is in contact with the solid phase surface of the substrate is applied to the solid surface surface of the substrate. Is a target measurement method that measures from the opposite side.
[2] The target is modified with the fluorescent molecule, and the target is modified with the fluorescent molecule.
The target measurement method according to [1], wherein the target is bound to the trapping molecule immobilized on the solid phase surface to obtain the conjugate.
[3] The capture molecule is modified with the fluorescent molecule, and the capture molecule is modified with the fluorescent molecule.
The target measurement method according to [1], wherein the capture molecule is bound to the target immobilized on the solid phase surface to obtain the conjugate.
[4] The capture molecule is modified with the fluorescent molecule, and the capture molecule is modified with the fluorescent molecule.
The target measurement method according to [1], wherein the target is bound to the trapping molecule immobilized on the solid phase surface to obtain the conjugate.
[5] The target and one of the trapping molecules immobilized on the solid phase surface are supplied with the other of the target and the trapping molecule and the fluorescent molecule to obtain the conjugate. Obtain, the target measurement method according to [1].
[6] The target is a nucleic acid having a specific nucleic acid sequence.
The capture molecule is a detection probe having a sequence complementary to the specific nucleic acid sequence.
The target measurement method according to any one of [1] to [5], wherein the target is hybridized with the detection probe to obtain the conjugate.
[7] A target measurement device used when measuring a target contained in a sample.
A substrate in which either one of the target and a capture molecule specifically bound to the target is immobilized on a solid phase surface, and a substrate.
A container to which an excitation light that excites a fluorescent molecule that modifies a bond between the target and the capture molecule or an absorbent substance that absorbs the fluorescence emitted from the fluorescent molecule is added, that is, the target and the capture molecule. A container capable of holding a solution containing either the other and the absorbent substance in contact with the solid phase surface of the substrate.
A device for target measurement equipped with.
[8] The trapping molecule is immobilized on the solid phase surface.
The target measurement device according to [7], wherein the container is supplied with a solution containing the target modified with the fluorescent molecule.
[9] The target is immobilized on the solid surface.
The target measurement device according to [7], wherein the container is supplied with a solution containing the capture molecule modified with the fluorescent molecule.
[10] The capture molecule modified with the fluorescent molecule is immobilized on the solid phase surface.
The target measurement device according to [7], wherein a solution containing the target is supplied to the container.
[11] Either the target or the capture molecule is immobilized on the solid phase surface.
The target according to [7], wherein the container is supplied with a solution containing any one of the target and the capture molecule and the fluorescent molecule that binds to the conjugate of the target and the capture molecule. Measurement device.
[12] The target is a target having a specific nucleic acid sequence.
The target measurement device according to any one of [7] to [11], wherein the capture molecule is a detection probe having a sequence complementary to the specific nucleic acid sequence.
[13] The target measurement device according to any one of [7] to [12], and
A fluorescence reader that measures the amount of fluorescence from the target measurement device,
Has a target measuring device.
[14] A target measurement kit used when measuring a target contained in a sample.
A substrate in which one of the target and a capture molecule that specifically binds to the target is immobilized on a solid phase surface, and a substrate.
A container capable of holding a solution containing either one of the target and the trapping molecule in contact with the solid phase surface of the substrate.
Excitation light that excites a fluorescent molecule that modifies the conjugate of the target and the capture molecule, or an absorbent substance that absorbs the fluorescence emitted from the fluorescent molecule.
Target measurement kit including.
[15] The target measurement kit according to [14], wherein the absorbent substance is preliminarily added to the container.
[16] The trapping molecule is immobilized on the solid phase surface.
The target measurement kit according to [14] or [15], wherein the container holds a solution containing the target modified with the fluorescent molecule and the absorbent substance.
[17] The target is immobilized on the solid surface.
The target measurement kit according to [14] or [15], wherein the container holds a solution containing the capture molecule modified with the fluorescent molecule and the absorbent substance.
[18] The capture molecule modified with the fluorescent molecule is immobilized on the solid phase surface.
The target measurement kit according to [14] or [15], wherein the container holds a solution containing the target and the light-absorbing substance.
[19] Either the target or the capture molecule is immobilized on the solid phase surface.
The container holds a solution containing either one of the target and the capture molecule, the fluorescent molecule that binds to the conjugate of the target and the capture molecule, and the absorbance substance [14. ] Or the target measurement kit according to [15].
[20] The target is a target having a specific nucleic acid sequence.
The target measurement kit according to any one of [14] to [19], wherein the capture molecule is a detection probe having a sequence complementary to the specific nucleic acid sequence.
［１］ サンプルに含まれるターゲットを計測するターゲット計測方法であって、
蛍光分子で修飾された、前記ターゲットと前記ターゲットに特異的に結合する捕捉分子との結合物が設けられた基板の固相面に、前記蛍光分子を励起する励起光、又は前記蛍光分子から発せられる蛍光を吸収する吸光物質を含む溶液が接触している状態で、前記基板の前記固相面とは反対側から前記励起光を照射して得られる蛍光を、前記基板の前記固相面とは反対側から計測するターゲット計測方法。
［２］ 前記ターゲットを前記蛍光分子で修飾し、
前記固相面に固定化されている前記捕捉分子に前記ターゲットを結合させて前記結合物を得る、［１］に記載のターゲット計測方法。
［３］ 前記捕捉分子を前記蛍光分子で修飾し、
前記固相面に固定化されている前記ターゲットに前記捕捉分子を結合させて前記結合物を得る、［１］に記載のターゲット計測方法。
［４］ 前記捕捉分子を前記蛍光分子で修飾し、
前記固相面に固定化されている前記捕捉分子に、前記ターゲットを結合させて前記結合物を得る、［１］に記載のターゲット計測方法。
［５］ 前記固相面に固定化されている、前記ターゲット及び前記捕捉分子のいずれか一方に対し、前記ターゲット及び前記捕捉分子のいずれか他方と前記蛍光分子とを供給して前記結合物を得る、［１］に記載のターゲット計測方法。
［６］ 前記ターゲットは、特定の核酸配列を有する核酸であり、
前記捕捉分子は、前記特定の核酸配列と相補的な配列を有する検出プローブであり、
前記ターゲットを前記検出プローブにハイブリダイズ反応させて前記結合物を得る、［１］～[５]のいずれか１項に記載のターゲット計測方法。
［７］ サンプルに含まれるターゲットを計測する際に用いられるターゲット計測用デバイスであって、
前記ターゲットと前記ターゲットに特異的に結合する捕捉分子とのいずれか一方が固相面に固定化される基板と、
前記ターゲットと前記捕捉分子との結合物を修飾する蛍光分子を励起する励起光、又は前記蛍光分子から発せられる蛍光を吸収する吸光物質が添加された容器であって、前記ターゲット及び前記捕捉分子のいずれか他方と、前記吸光物質とを含む溶液を、前記基板の前記固相面に接触させた状態で保持可能な容器と、
を備えたターゲット計測用デバイス。
［８］ 前記固相面には、前記捕捉分子が固定化されており、
前記容器には、前記蛍光分子で修飾された前記ターゲットを含む溶液が供給される、［７］に記載のターゲット計測用デバイス。
［９］ 前記固相面には、前記ターゲットが固定化されており、
前記容器には、前記蛍光分子で修飾された前記捕捉分子を含む溶液が供給される、［７］に記載のターゲット計測用デバイス。
［１０］ 前記固相面には、前記蛍光分子で修飾されている、前記捕捉分子が固定化されており、
前記容器には、前記ターゲットを含む溶液が供給される、［７］に記載のターゲット計測用デバイス。
［１１］ 前記固相面には、前記ターゲット及び前記捕捉分子のいずれか一方が固定化されており、
前記容器には、前記ターゲット及び前記捕捉分子のいずれか他方と、前記ターゲットと前記捕捉分子との結合体に結合する前記蛍光分子と、を含む溶液が供給される、［７］に記載のターゲット計測用デバイス。
［１２］ 前記ターゲットは、特定の核酸配列を有するターゲットであり、
前記捕捉分子は、前記特定の核酸配列に相補的な配列を有する検出プローブである、［７］～［１１］のいずれか１項に記載のターゲット計測用デバイス。
［１３］ ［７］～［１２］のいずれか１項に記載のターゲット計測用デバイスと、
前記ターゲット計測用デバイスからの蛍光量を測定する蛍光読取装置と、
を有する、ターゲット計測装置。
［１４］ サンプルに含まれるターゲットを計測する際に用いられるターゲット計測用キットであって、
前記ターゲットと前記ターゲットに特異的に結合する捕捉分子とのいずれか一方が固相面に固定化された基板と、
前記ターゲット及び前記捕捉分子のいずれか他方を含む溶液を、前記基板の前記固相面に接触させた状態で保持可能な容器と、
前記ターゲットと前記捕捉分子との結合物を修飾する蛍光分子を励起する励起光、又は前記蛍光分子から発せられる蛍光を吸収する吸光物質と、
を含むターゲット計測用キット。
［１５］ 前記吸光物質は、前記容器に予め添加されている、［１４］に記載のターゲット計測用キット。
［１６］ 前記固相面には、前記捕捉分子が固定化されており、
前記容器には、前記蛍光分子で修飾された前記ターゲットと、前記吸光物質とを含む溶液が保持される、［１４］又は［１５］に記載のターゲット計測用キット。
［１７］ 前記固相面には、前記ターゲットが固定化されており、
前記容器には、前記蛍光分子で修飾された前記捕捉分子と、前記吸光物質とを含む溶液が保持される、［１４］又は［１５］に記載のターゲット計測用キット。
［１８］ 前記固相面には、前記蛍光分子で修飾されている前記捕捉分子が固定化されており、
前記容器には、前記ターゲットと前記吸光物質とを含む溶液が保持される、［１４］又は［１５］に記載のターゲット計測用キット。
［１９］ 前記固相面には、前記ターゲット及び前記捕捉分子のいずれか一方が固定化されており、
前記容器には、前記ターゲット及び前記捕捉分子のいずれか他方と、前記ターゲットと前記捕捉分子との結合体に結合する前記蛍光分子と、前記吸光物質と、を含む溶液が保持される、［１４］又は［１５］に記載のターゲット計測用キット。
［２０］ 前記ターゲットは、特定の核酸配列を有するターゲットであり、
前記捕捉分子は、前記特定の核酸配列に相補的な配列を有する検出プローブである、［１４］～［１９］のいずれか１項に記載のターゲット計測用キット。 In order to achieve the above object, the present invention has adopted the following configuration.
[1] A target measurement method for measuring a target included in a sample.
The excitation light that excites the fluorescent molecule or the fluorescent molecule emits light on the solid phase surface of the substrate provided with the conjugate of the target and the capture molecule that specifically binds to the target, which is modified with the fluorescent molecule. The fluorescence obtained by irradiating the excitation light from the side opposite to the solid phase surface of the substrate in a state where the solution containing the absorbent substance that absorbs the fluorescence is in contact with the solid phase surface of the substrate is applied to the solid surface surface of the substrate. Is a target measurement method that measures from the opposite side.
[2] The target is modified with the fluorescent molecule, and the target is modified with the fluorescent molecule.
The target measurement method according to [1], wherein the target is bound to the trapping molecule immobilized on the solid phase surface to obtain the conjugate.
[3] The capture molecule is modified with the fluorescent molecule, and the capture molecule is modified with the fluorescent molecule.
The target measurement method according to [1], wherein the capture molecule is bound to the target immobilized on the solid phase surface to obtain the conjugate.
[4] The capture molecule is modified with the fluorescent molecule, and the capture molecule is modified with the fluorescent molecule.
The target measurement method according to [1], wherein the target is bound to the trapping molecule immobilized on the solid phase surface to obtain the conjugate.
[5] The target and one of the trapping molecules immobilized on the solid phase surface are supplied with the other of the target and the trapping molecule and the fluorescent molecule to obtain the conjugate. Obtain, the target measurement method according to [1].
[6] The target is a nucleic acid having a specific nucleic acid sequence.
The capture molecule is a detection probe having a sequence complementary to the specific nucleic acid sequence.
The target measurement method according to any one of [1] to [5], wherein the target is hybridized with the detection probe to obtain the conjugate.
[7] A target measurement device used when measuring a target contained in a sample.
A substrate in which either one of the target and a capture molecule specifically bound to the target is immobilized on a solid phase surface, and a substrate.
A container to which an excitation light that excites a fluorescent molecule that modifies a bond between the target and the capture molecule or an absorbent substance that absorbs the fluorescence emitted from the fluorescent molecule is added, that is, the target and the capture molecule. A container capable of holding a solution containing either the other and the absorbent substance in contact with the solid phase surface of the substrate.
A device for target measurement equipped with.
[8] The trapping molecule is immobilized on the solid phase surface.
The target measurement device according to [7], wherein the container is supplied with a solution containing the target modified with the fluorescent molecule.
[9] The target is immobilized on the solid surface.
The target measurement device according to [7], wherein the container is supplied with a solution containing the capture molecule modified with the fluorescent molecule.
[10] The capture molecule modified with the fluorescent molecule is immobilized on the solid phase surface.
The target measurement device according to [7], wherein a solution containing the target is supplied to the container.
[11] Either the target or the capture molecule is immobilized on the solid phase surface.
The target according to [7], wherein the container is supplied with a solution containing any one of the target and the capture molecule and the fluorescent molecule that binds to the conjugate of the target and the capture molecule. Measurement device.
[12] The target is a target having a specific nucleic acid sequence.
The target measurement device according to any one of [7] to [11], wherein the capture molecule is a detection probe having a sequence complementary to the specific nucleic acid sequence.
[13] The target measurement device according to any one of [7] to [12], and
A fluorescence reader that measures the amount of fluorescence from the target measurement device,
Has a target measuring device.
[14] A target measurement kit used when measuring a target contained in a sample.
A substrate in which one of the target and a capture molecule that specifically binds to the target is immobilized on a solid phase surface, and a substrate.
A container capable of holding a solution containing either one of the target and the trapping molecule in contact with the solid phase surface of the substrate.
Excitation light that excites a fluorescent molecule that modifies the conjugate of the target and the capture molecule, or an absorbent substance that absorbs the fluorescence emitted from the fluorescent molecule.
Target measurement kit including.
[15] The target measurement kit according to [14], wherein the absorbent substance is preliminarily added to the container.
[16] The trapping molecule is immobilized on the solid phase surface.
The target measurement kit according to [14] or [15], wherein the container holds a solution containing the target modified with the fluorescent molecule and the absorbent substance.
[17] The target is immobilized on the solid surface.
The target measurement kit according to [14] or [15], wherein the container holds a solution containing the capture molecule modified with the fluorescent molecule and the absorbent substance.
[18] The capture molecule modified with the fluorescent molecule is immobilized on the solid phase surface.
The target measurement kit according to [14] or [15], wherein the container holds a solution containing the target and the light-absorbing substance.
[19] Either the target or the capture molecule is immobilized on the solid phase surface.
The container holds a solution containing either one of the target and the capture molecule, the fluorescent molecule that binds to the conjugate of the target and the capture molecule, and the absorbance substance [14. ] Or the target measurement kit according to [15].
[20] The target is a target having a specific nucleic acid sequence.
The target measurement kit according to any one of [14] to [19], wherein the capture molecule is a detection probe having a sequence complementary to the specific nucleic acid sequence.
本発明のターゲット計測方法、ターゲット計測用デバイス、ターゲット計測装置、及びターゲット計測用キットによれば、サンプルに含まれるターゲットの計測精度を従来よりも向上させることができるという効果がある。
According to the target measurement method, the target measurement device, the target measurement device, and the target measurement kit of the present invention, there is an effect that the measurement accuracy of the target included in the sample can be improved as compared with the conventional case.
以下、図面を参照して本発明の実施形態によるターゲット計測方法、ターゲット計測用デバイス、ターゲット計測装置、及びターゲット計測用キットについて詳細に説明する。以下では、まず本発明の実施形態の概要について説明し、続いて本発明の実施形態の詳細について説明する。
Hereinafter, the target measurement method, the target measurement device, the target measurement device, and the target measurement kit according to the embodiment of the present invention will be described in detail with reference to the drawings. Hereinafter, the outline of the embodiment of the present invention will be described first, and then the details of the embodiment of the present invention will be described.
〔概要〕
本発明の実施形態は、サンプルに含まれるターゲットの計測精度を従来よりも向上させることができるようにするものである。上述した非特許文献１に開示された方法は、ＤＮＡサンプルに蛍光修飾プライマーを用いてＰＣＲを行って得られた蛍光修飾されたＰＣＲ産物を、ＤＮＡマイクロアレイに添加してハイブリダイズ反応させ、サンプル中のターゲットを検出するものである。上述した非特許文献２に開示された方法は、ターゲットをマイクロアレイに固定化したものと蛍光修飾された蛍光プローブとをハイブリダイズ反応させ、サンプル中のターゲットを検出するものである。 〔Overview〕
The embodiment of the present invention makes it possible to improve the measurement accuracy of the target included in the sample as compared with the conventional case. In the method disclosed in Non-Patent Document 1 described above, a fluorescence-modified PCR product obtained by performing PCR on a DNA sample using a fluorescence-modifying primer is added to a DNA microarray and hybridized, and the sample is mixed. It detects the target of. The method disclosed inNon-Patent Document 2 described above is to detect a target in a sample by hybridizing and reacting a target immobilized on a microarray with a fluorescence-modified fluorescent probe.
本発明の実施形態は、サンプルに含まれるターゲットの計測精度を従来よりも向上させることができるようにするものである。上述した非特許文献１に開示された方法は、ＤＮＡサンプルに蛍光修飾プライマーを用いてＰＣＲを行って得られた蛍光修飾されたＰＣＲ産物を、ＤＮＡマイクロアレイに添加してハイブリダイズ反応させ、サンプル中のターゲットを検出するものである。上述した非特許文献２に開示された方法は、ターゲットをマイクロアレイに固定化したものと蛍光修飾された蛍光プローブとをハイブリダイズ反応させ、サンプル中のターゲットを検出するものである。 〔Overview〕
The embodiment of the present invention makes it possible to improve the measurement accuracy of the target included in the sample as compared with the conventional case. In the method disclosed in Non-Patent Document 1 described above, a fluorescence-modified PCR product obtained by performing PCR on a DNA sample using a fluorescence-modifying primer is added to a DNA microarray and hybridized, and the sample is mixed. It detects the target of. The method disclosed in
上述した特許文献１に開示された方法は、検出プローブとして、蛍光分子が付加された蛍光プローブと、蛍光分子の蛍光を消光する消光分子が付加された消光プローブとが設けられた核酸配列計測デバイス（ＤＮＡマイクロアレイ）を用いてターゲットを計測するものである。この方法では、ターゲットに対する蛍光分子の付加、及びＤＮＡマイクロアレイの洗浄（捕集されていないターゲット等を除去するための洗浄）を行うことなくターゲットを計測することが可能である。この方法では、ＤＮＡマイクロアレイを用いて特定の核酸の有無や量を計測する核酸配列計測デバイスにおいて、ターゲットが存在しないときは、互いに独立したドナー蛍光プローブ及び消光プローブが、結合部を介して結合が維持されて消光分子により蛍光分子の蛍光が消光されている。ターゲットが供給された場合には、検出部にターゲットが結合して、結合部を介するドナー蛍光プローブと消光プローブとの結合が解消され、消光分子がドナー蛍光分子から離れることによりドナー蛍光分子が蛍光を呈する。この核酸配列計測デバイスを用いることにより、サンプルに含まれるターゲットを計測することができる。
The method disclosed in Patent Document 1 described above is a nucleic acid sequence measurement device provided with a fluorescent probe to which a fluorescent molecule is added and a quenching probe to which a quenching molecule for quenching the fluorescence of the fluorescent molecule is added as a detection probe. (DNA microarray) is used to measure the target. In this method, it is possible to measure the target without adding fluorescent molecules to the target and washing the DNA microarray (washing for removing uncollected targets and the like). In this method, in a nucleic acid sequence measuring device that measures the presence or absence or amount of a specific nucleic acid using a DNA microarray, when a target is not present, donor fluorescent probes and quenching probes that are independent of each other are bound via a binding portion. It is maintained and the fluorescence of the fluorescent molecule is quenched by the quenching molecule. When the target is supplied, the target binds to the detection part, the bond between the donor fluorescent probe and the quenching probe via the binding part is broken, and the quenching molecule separates from the donor fluorescent molecule, so that the donor fluorescent molecule fluoresces. Present. By using this nucleic acid sequence measuring device, the target contained in the sample can be measured.
しかしながら、上述した非特許文献１の核酸配列計測方法では、捕集されていないターゲットを除去するための洗浄操作が必要であり、洗浄操作により反応したターゲットが剥離し、ターゲットの定量性の低下、検出下限が悪化する可能性がある。非特許文献２の核酸配列計測方法も同様に、捕集されていないプローブを除去するための洗浄操作が必要であり、洗浄操作により反応したプローブが剥離し、ターゲットの定量性の低下、検出下限が悪化する可能性がある。
また、非特許文献１及び非特許文献２の核酸配列計測方法では、当該方法に必要とされる洗浄操作によりマイクロアレイ上で隣接するウェルの間でサンプルが混入し、正確にターゲットが検出できない可能性がある。 However, the nucleic acid sequence measurement method of Non-Patent Document 1 described above requires a washing operation for removing a target that has not been collected, and the reaction target is exfoliated by the washing operation, resulting in a decrease in the quantitativeness of the target. The lower limit of detection may worsen. Similarly, the nucleic acid sequence measurement method ofNon-Patent Document 2 requires a washing operation for removing uncollected probes, and the reacted probe is peeled off by the washing operation, the quantitativeness of the target is lowered, and the lower limit of detection is reached. May get worse.
Further, in the nucleic acid sequence measurement methods of Non-Patent Document 1 andNon-Patent Document 2, there is a possibility that the sample is mixed between adjacent wells on the microarray due to the washing operation required for the method, and the target cannot be detected accurately. There is.
また、非特許文献１及び非特許文献２の核酸配列計測方法では、当該方法に必要とされる洗浄操作によりマイクロアレイ上で隣接するウェルの間でサンプルが混入し、正確にターゲットが検出できない可能性がある。 However, the nucleic acid sequence measurement method of Non-Patent Document 1 described above requires a washing operation for removing a target that has not been collected, and the reaction target is exfoliated by the washing operation, resulting in a decrease in the quantitativeness of the target. The lower limit of detection may worsen. Similarly, the nucleic acid sequence measurement method of
Further, in the nucleic acid sequence measurement methods of Non-Patent Document 1 and
上述した特許文献１では、ＤＮＡマイクロアレイの洗浄（捕集されていないターゲット等を除去するための洗浄）を行うことなくターゲットを計測することが可能である。しかしながら、ターゲットの調製として生体や微生物などを含む検体から抽出した核酸サンプルには検体由来のタンパク質や糖類などの残留分子が含まれていることが多い。そのため核酸サンプルの溶液は蛍光画像を取得する際に蛍光を発しており、背景光の光量が高くなる要因となっており、核酸配列計測デバイスに供給するサンプルの溶液からオフセット光が発せられてしまう。このようなオフセット光は、ノイズとなることから計測精度を悪化させてしまう。例えば、ターゲットが僅かである場合には、核酸配列計測デバイスに供給するサンプルの溶液から発せられる蛍光も微弱なものとなるところ、この微弱な蛍光が、オフセット光に埋もれてしまうとターゲットの計測を行うことができない。
In Patent Document 1 described above, it is possible to measure a target without cleaning the DNA microarray (cleaning for removing a target or the like that has not been collected). However, nucleic acid samples extracted from a sample containing a living body or a microorganism as a target preparation often contain residual molecules such as proteins and sugars derived from the sample. Therefore, the nucleic acid sample solution emits fluorescence when acquiring a fluorescent image, which causes an increase in the amount of background light, and offset light is emitted from the sample solution supplied to the nucleic acid sequence measurement device. .. Such offset light becomes noise and deteriorates the measurement accuracy. For example, when the number of targets is small, the fluorescence emitted from the solution of the sample supplied to the nucleic acid sequence measurement device is also weak, and when this weak fluorescence is buried in the offset light, the target is measured. I can't do it.
〔実施形態〕
本実施形態のターゲット計測方法は、蛍光分子で修飾された、ターゲットと前記ターゲットに特異的に結合する捕捉分子（以下、単に「捕捉分子」とも称することもある）との結合物が設けられた基板の固相面に、前記蛍光分子を励起する励起光、又は前記蛍光分子から発せられる蛍光を吸収する吸光物質（以下、単に「吸光物質」と称することもある）を含む溶液が接触している状態で、前記基板の固相面とは反対側から前記蛍光分子を励起する励起光を照射して得られる蛍光を、前記基板の前記固相面とは反対側から計測するようにしている。これにより、サンプルに含まれるターゲットの計測精度を従来よりも向上させることができる。また、ＤＮＡマイクロアレイ等の基板の洗浄操作を不要にし、洗浄操作に基づく定量性低下や検出下限の悪化の影響を除外することができる。また、検体由来のサンプル溶液の蛍光を低減し、微弱光を計測することができる。 [Embodiment]
In the target measurement method of the present embodiment, a conjugate of the target and a capture molecule that specifically binds to the target (hereinafter, may be simply referred to as “capture molecule”) modified with a fluorescent molecule is provided. A solution containing an excitation light that excites the fluorescent molecule or an absorbent substance that absorbs the fluorescence emitted from the fluorescent molecule (hereinafter, may be simply referred to as “absorbent substance”) is in contact with the solid phase surface of the substrate. In this state, the fluorescence obtained by irradiating the excitation light that excites the fluorescent molecule from the side opposite to the solid surface surface of the substrate is measured from the side opposite to the solid phase surface of the substrate. .. As a result, the measurement accuracy of the target included in the sample can be improved as compared with the conventional case. In addition, it is possible to eliminate the need for a substrate cleaning operation such as a DNA microarray, and to eliminate the effects of a decrease in quantification and a deterioration in the lower limit of detection due to the cleaning operation. In addition, it is possible to reduce the fluorescence of the sample solution derived from the sample and measure weak light.
本実施形態のターゲット計測方法は、蛍光分子で修飾された、ターゲットと前記ターゲットに特異的に結合する捕捉分子（以下、単に「捕捉分子」とも称することもある）との結合物が設けられた基板の固相面に、前記蛍光分子を励起する励起光、又は前記蛍光分子から発せられる蛍光を吸収する吸光物質（以下、単に「吸光物質」と称することもある）を含む溶液が接触している状態で、前記基板の固相面とは反対側から前記蛍光分子を励起する励起光を照射して得られる蛍光を、前記基板の前記固相面とは反対側から計測するようにしている。これにより、サンプルに含まれるターゲットの計測精度を従来よりも向上させることができる。また、ＤＮＡマイクロアレイ等の基板の洗浄操作を不要にし、洗浄操作に基づく定量性低下や検出下限の悪化の影響を除外することができる。また、検体由来のサンプル溶液の蛍光を低減し、微弱光を計測することができる。 [Embodiment]
In the target measurement method of the present embodiment, a conjugate of the target and a capture molecule that specifically binds to the target (hereinafter, may be simply referred to as “capture molecule”) modified with a fluorescent molecule is provided. A solution containing an excitation light that excites the fluorescent molecule or an absorbent substance that absorbs the fluorescence emitted from the fluorescent molecule (hereinafter, may be simply referred to as “absorbent substance”) is in contact with the solid phase surface of the substrate. In this state, the fluorescence obtained by irradiating the excitation light that excites the fluorescent molecule from the side opposite to the solid surface surface of the substrate is measured from the side opposite to the solid phase surface of the substrate. .. As a result, the measurement accuracy of the target included in the sample can be improved as compared with the conventional case. In addition, it is possible to eliminate the need for a substrate cleaning operation such as a DNA microarray, and to eliminate the effects of a decrease in quantification and a deterioration in the lower limit of detection due to the cleaning operation. In addition, it is possible to reduce the fluorescence of the sample solution derived from the sample and measure weak light.
本実施形態のターゲット計測方法において、サンプルに含まれるターゲットを計測するターゲット計測方法であって、蛍光分子で修飾された、前記ターゲットと前記ターゲットに特異的に結合する捕捉分子の結合物を得る方法としては、例えば、前記ターゲットを前記蛍光分子で修飾し、前記固相面に固定化されている前記捕捉分子に前記ターゲットを結合させて前記結合物を得る方法；前記捕捉分子を前記蛍光分子で修飾し、前記固相面に固定化されている前記ターゲットに前記捕捉分子を結合させて前記結合物を得る方法；前記捕捉分子を前記蛍光分子で修飾し、固相面に固定化されている前記捕捉分子に、前記ターゲットを結合させて前記結合物を得る方法；前記固相面に固定化されている、前記ターゲット及び前記捕捉分子のいずれか一方に対し、前記ターゲット及び前記捕捉分子のいずれか他方と前記蛍光分子とを供給して前記結合物を得る方法等が挙げられる。
In the target measurement method of the present embodiment, which is a target measurement method for measuring a target contained in a sample, a method for obtaining a conjugate of the target and a capture molecule specifically bound to the target, which is modified with a fluorescent molecule. For example, a method of modifying the target with the fluorescent molecule and binding the target to the capture molecule immobilized on the solid phase surface to obtain the conjugate; the capture molecule is used with the fluorescent molecule. A method of modifying and binding the capture molecule to the target immobilized on the solid surface to obtain the conjugate; the capture molecule is modified with the fluorescent molecule and immobilized on the solid surface. A method of binding the target to the trapping molecule to obtain the conjugate; either the target or the trapping molecule, which is immobilized on the solid phase surface, with respect to either the target or the trapping molecule. Examples thereof include a method of supplying the other and the fluorescent molecule to obtain the conjugate.
前記固相面に固定化されている、前記蛍光分子で修飾された前記捕捉分子に、前記ターゲットを結合させて前記結合物を得る方法としては、例えば、蛍光分子で修飾された捕捉分子と、消光物質で修飾された前記捕捉分子と特異的に結合する消光分子とを、前記捕捉分子を修飾している蛍光分子が、前記消光分子を修飾している消光物質によって消光されるように、固相面に固定化し、ターゲットが存在しないときは、蛍光分子を励起光で励起しても蛍光が発生しないようにし、ターゲットが存在するときに、ターゲットを、前記捕捉分子と結合させて結合物を得る方法が挙げられる。ターゲットが前記捕捉分子に結合すると、前記消光分子を修飾している消光物質が、前記捕捉分子を修飾している蛍光分子から離れることによって、固相面に固定化された前記捕捉分子から蛍光が発生する。
As a method for obtaining the conjugate by binding the target to the capture molecule modified with the fluorescent molecule, which is immobilized on the solid phase surface, for example, a capture molecule modified with the fluorescent molecule and the capture molecule are used. The quenching molecule that specifically binds to the quenching molecule modified with the quenching substance is solidified so that the fluorescent molecule modifying the capturing molecule is extinguished by the quenching substance modifying the quenching molecule. It is immobilized on the phase surface so that when the target does not exist, fluorescence does not occur even if the fluorescent molecule is excited by excitation light, and when the target exists, the target is bound to the capture molecule to form a conjugate. There is a way to get it. When the target binds to the capture molecule, the quenching substance modifying the quenching molecule separates from the fluorescent molecule modifying the capture molecule, so that fluorescence is emitted from the capture molecule immobilized on the solid phase surface. Occur.
前記固相面に固定化されている、前記ターゲット及び前記捕捉分子のいずれか一方に対し、前記ターゲット及び前記捕捉分子のいずれか他方と前記蛍光分子とを供給して前記結合物を得る方法としては、例えば、前記固相面に、特定の核酸配列を有する核酸及び前記核酸の特定の核酸配列と相補的な核酸配列を有する核酸プローブのいずれか一方を固定化し、前記核酸及び前記核酸プローブのいずれか他方と、蛍光分子で修飾した、前記核酸と前記核酸プローブの結合体とに結合するインターカレーターとを固相面に供給して結合物を得る方法が挙げられる。
As a method for obtaining the conjugate by supplying either the target or the capture molecule and the fluorescent molecule to either the target or the capture molecule immobilized on the solid phase surface. For example, one of a nucleic acid having a specific nucleic acid sequence and a nucleic acid probe having a nucleic acid sequence complementary to the specific nucleic acid sequence of the nucleic acid is immobilized on the solid phase surface of the nucleic acid and the nucleic acid probe. Examples thereof include a method of supplying either one and an intercalator modified with a fluorescent molecule to bind to the nucleic acid and the conjugate of the nucleic acid probe to the solid phase surface to obtain a conjugate.
ターゲットしては、サンプル中の検出の対象となるものであれば、特に制限はないが、例えば、ＤＮＡ、ＲＮＡ等の核酸、ペプチド、タンパク質等が挙げられる。ターゲットに特異的に結合する捕捉分子としては、核酸とハイブリダイズする検出プローブ、ペプチド、タンパク質等の抗原と特異的に結合する抗体若しくは抗体フラグメント、核酸と特異的に結合するアプタマー等が挙げられる。前記抗体としては、ポリクローナル抗体、モノクローナル抗体のいずれも用いることができるが、モノクローナル抗体が好ましい。抗体フラグメントとしては、例えば、Ｆ（ａｂ’）２、Ｆ（ａｂ）２、Ｆａｂ’、Ｆａｂ、Ｆｖ、ｓｃＦｖ、これらの変異体、抗体部分を含む融合タンパク質又は融合ペプチド等が挙げられる。また、ターゲットが抗体又は抗体フラグメントであって、捕捉分子が該抗体又は抗体フラグメントと特異的に結合するペプチド、タンパク質等の抗原であってもよい。
The target is not particularly limited as long as it is a target to be detected in the sample, and examples thereof include nucleic acids such as DNA and RNA, peptides, and proteins. Examples of the capture molecule that specifically binds to the target include a detection probe that hybridizes with nucleic acid, an antibody or antibody fragment that specifically binds to an antigen such as a peptide or protein, and an aptamer that specifically binds to nucleic acid. As the antibody, either a polyclonal antibody or a monoclonal antibody can be used, but a monoclonal antibody is preferable. Examples of the antibody fragment include F (ab') 2 , F (ab) 2 , Fab', Fab, Fv, scFv, variants thereof, fusion proteins or fusion peptides containing an antibody moiety, and the like. Further, the target may be an antibody or an antibody fragment, and the capture molecule may be an antigen such as a peptide or protein that specifically binds to the antibody or antibody fragment.
ターゲットと前記ターゲットに特異的に結合する捕捉分子の組み合わせとしては、例えば、ターゲットが特定の核酸配列を有する核酸であって、捕捉分子が前記特定の核酸配列と相補的な配列を有する検出プローブである組み合わせ、ターゲットが抗原であって、捕捉分子が前記抗原と特異的に結合する抗体又は抗体フラグメントである組み合わせ等が挙げられる。ターゲットが特定の核酸配列を有する核酸であって、捕捉分子が前記特定の核酸配列と相補的な配列を有する検出プローブである場合、ターゲットである核酸は捕捉分子である検出プローブにハイブリダイズ反応により結合する。
The combination of the target and the capture molecule that specifically binds to the target is, for example, a detection probe in which the target is a nucleic acid having a specific nucleic acid sequence and the capture molecule has a sequence complementary to the specific nucleic acid sequence. Examples thereof include a combination in which the target is an antigen and the capture molecule is an antibody or an antibody fragment that specifically binds to the antigen. When the target is a nucleic acid having a specific nucleic acid sequence and the capture molecule is a detection probe having a sequence complementary to the specific nucleic acid sequence, the target nucleic acid is hybridized to the detection probe which is the capture molecule. Join.
図１に、前記ターゲットを前記蛍光分子で修飾し、前記固相面に固定化されている前記捕捉分子に前記ターゲットを結合させて前記結合物を得る方法の具体例を示す。図１では、ターゲット３が特定の核酸配列を有するＤＮＡであって、該ＤＮＡが蛍光分子４で修飾されており、捕捉分子が、前記ターゲット３の特定の核酸配列と相補的な核酸配列である検出配列２を有するＤＮＡプローブ１である。前記ＤＮＡプローブ１は基板であるＤＮＡマイクロアレイ５にリンカー２１を介して固定化されている。蛍光分子４で修飾されたターゲット３は、ＤＮＡマイクロアレイ５に固定化されたＤＮＡプローブ１とハイブリダイズ反応により結合し、ターゲット３を修飾している蛍光分子４を励起光で励起することにより蛍光が放射される。
FIG. 1 shows a specific example of a method of modifying the target with the fluorescent molecule and binding the target to the capture molecule immobilized on the solid phase surface to obtain the conjugate. In FIG. 1, target 3 is DNA having a specific nucleic acid sequence, the DNA is modified with fluorescent molecule 4, and the capture molecule is a nucleic acid sequence complementary to the specific nucleic acid sequence of target 3. It is a DNA probe 1 having a detection sequence 2. The DNA probe 1 is immobilized on a DNA microarray 5 which is a substrate via a linker 21. The target 3 modified with the fluorescent molecule 4 binds to the DNA probe 1 immobilized on the DNA microarray 5 by a hybridization reaction, and the fluorescent molecule 4 modifying the target 3 is excited by excitation light to cause fluorescence. Be radiated.
図２に、前記捕捉分子を前記蛍光分子で修飾し、前記固相面に固定化されている前記ターゲットに前記捕捉分子を結合させて前記結合物を得る方法の具体例を示す。図２では、ターゲット３が特定の核酸配列を有するＤＮＡであって、捕捉分子が、前記ターゲット３の特定の核酸配列と相補的な核酸配列である検出配列２を有するＤＮＡプローブ１である。ＤＮＡプローブ１は蛍光分子４で修飾されており、ターゲット３が基板であるＤＮＡマイクロアレイ５に固定化されている。蛍光分子４で修飾されたＤＮＡプローブ１は、ＤＮＡマイクロアレイ５に固定化されたターゲット３とハイブリダイズ反応により結合し、ＤＮＡプローブ１を修飾している蛍光分子４を励起光で励起することにより蛍光が放射される。
FIG. 2 shows a specific example of a method of modifying the capture molecule with the fluorescent molecule and binding the capture molecule to the target immobilized on the solid phase surface to obtain the conjugate. In FIG. 2, the target 3 is a DNA having a specific nucleic acid sequence, and the capture molecule is a DNA probe 1 having a detection sequence 2 which is a nucleic acid sequence complementary to the specific nucleic acid sequence of the target 3. The DNA probe 1 is modified with the fluorescent molecule 4, and the target 3 is immobilized on the DNA microarray 5 which is a substrate. The DNA probe 1 modified with the fluorescent molecule 4 binds to the target 3 immobilized on the DNA microarray 5 by a hybridization reaction, and fluoresces by exciting the fluorescent molecule 4 modifying the DNA probe 1 with excitation light. Is radiated.
図３に、前記捕捉分子を前記蛍光分子で修飾し、前記固相面に固定化されている前記捕捉分子に、前記ターゲットを結合させて前記結合物を得る方法の具体例を示す。図３では、ターゲット３が特定の核酸配列を有するＤＮＡであって、捕捉分子が、蛍光分子４で修飾された、前記ターゲット３の特定の核酸配列と相補的な核酸配列である検出配列２を有するドナー蛍光プローブ６である。ドナー蛍光プローブ６は、基板であるＤＮＡマイクロアレイ５にリンカー２１を介して固定化されており、消光物質８で修飾されたドナー蛍光プローブ６の核酸配列と相補的な配列を有する消光プローブ７が、ドナー蛍光プローブ６と結合部２２でハイブリダイズするように、基板であるＤＮＡマイクロアレイ５に固定化されている。ターゲット３が存在しないときは、蛍光分子４が消光物質８によって消光された状態となっており、蛍光分子４を励起光で励起しても蛍光が発生しない。ターゲット３が存在する時は、ターゲット３が蛍光分子４で修飾されたドナー蛍光プローブ６の検出配列２にハイブリダイズ反応により結合し、前記消光プローブ７がドナー蛍光プローブ６から離れることによって、蛍光分子４で修飾されたドナー蛍光プローブ６を修飾している蛍光分子４を励起光で励起することにより蛍光が放射される。
FIG. 3 shows a specific example of a method of modifying the capture molecule with the fluorescent molecule and binding the target to the capture molecule immobilized on the solid phase surface to obtain the conjugate. In FIG. 3, the detection sequence 2 is a DNA in which the target 3 has a specific nucleic acid sequence and the capture molecule is a nucleic acid sequence complementary to the specific nucleic acid sequence of the target 3 modified with the fluorescent molecule 4. It is a donor fluorescent probe 6 having. The donor fluorescent probe 6 is immobilized on a DNA microarray 5 which is a substrate via a linker 21, and a quenching probe 7 having a sequence complementary to the nucleic acid sequence of the donor fluorescent probe 6 modified with the quenching substance 8 is provided. It is immobilized on the DNA microarray 5 which is a substrate so as to hybridize with the donor fluorescent probe 6 at the binding portion 22. When the target 3 does not exist, the fluorescent molecule 4 is in a state of being extinguished by the quenching substance 8, and no fluorescence is generated even if the fluorescent molecule 4 is excited by the excitation light. When the target 3 is present, the target 3 binds to the detection sequence 2 of the donor fluorescent probe 6 modified with the fluorescent molecule 4 by a hybrid reaction, and the extinguishing probe 7 separates from the donor fluorescent probe 6 to cause a fluorescent molecule. Fluorescence is emitted by exciting the fluorescent molecule 4 modifying the donor fluorescent probe 6 modified with 4 with excitation light.
なお、本発明において相補的であるとは、一方の核酸配列が、他方の核酸配列と２本鎖状態を形成することのできる核酸配列を持つことを意味し、必ずしも完全に相補的である必要はなく、いくつかのミスマッチ塩基対を含んでいてもよい。
Note that complementary in the present invention means that one nucleic acid sequence has a nucleic acid sequence capable of forming a double-stranded state with the other nucleic acid sequence, and is not necessarily completely complementary. May contain some mismatched base pairs.
本発明で用いられる蛍光分子としては、特定の励起光で励起され蛍光を発生する分子であれば特に制限はないが、 Ａｌｅｘａ Ｆｌｕｏｒ（登録商標）シリーズ、ＡＴＴＯシリーズ、Ｂｒｉｌｌｉａｎｔシリーズ、Ｃｈｒｏｍｅｏ（登録商標）シリーズ、Ｂａｃｔｅｒｉｏｃｈｌｏｒｉｎシリーズ、ＦＡＭ、ＴＡＭＲＡ、Ｃｙ色素シリーズ、ＦＩＴＣ、ＨｉＬｙｔｅ Ｆｌｕｏｒ（登録商標）シリーズ、Ｒｈｏｄａｍｉｎｅシリーズ、Ｔｉｄｅ Ｆｌｕｏｒ（登録商標）シリーズ、ｉＦｌｕｏｒ（登録商標）シリーズ、ＤＹ色素シリーズ等が挙げられる。
The fluorescent molecule used in the present invention is not particularly limited as long as it is a molecule that is excited by specific excitation light to generate fluorescence, but Alexa Fluor (registered trademark) series, ATTO series, Brilliant series, and Chromeo (registered trademark). Examples include the series, Bactiochlorin series, FAM, TAMRA, Cy dye series, FITC, HiLite Fluor (registered trademark) series, Rhodamine series, Tide Fluor (registered trademark) series, iFluor (registered trademark) series, and DY dye series.
本発明で用いられる基板としては、平面視したときの形状が矩形形状に形成された板状の石英、ガラス、シリコン、フッ化カルシウム及びサファイア等の単結晶、セラミックス、及び樹脂材料等を用いることができる。樹脂材料としては、光学的特性、化学的及び熱的安定性に優れたＣＯＰ（シクロオレフィンポリマー）、ＣＯＣ（環状オレフィンコポリマー）、ポリカーボネイト、アクリル系樹脂、ポリエチレン樹脂等が挙げられる。なお、基板を平面視したときの形状は任意の形状であってよい。本発明においては、基板の固相面とは反対側から蛍光分子を励起する励起光を照射して得られる蛍光を、前記基板の前記固相面とは反対側から計測するため、本発明で用いられる基板は、前記蛍光分子を励起する励起光及び前記励起光を照射して得られる蛍光を透過する材料を用いることが好ましい。
As the substrate used in the present invention, plate-shaped quartz, glass, silicon, single crystals such as calcium fluoride and sapphire, ceramics, resin materials and the like having a rectangular shape when viewed in a plan view are used. Can be done. Examples of the resin material include COP (cycloolefin polymer), COC (cyclic olefin copolymer), polycarbonate, acrylic resin, polyethylene resin and the like having excellent optical properties, chemical and thermal stability. The shape of the substrate when viewed in a plan view may be any shape. In the present invention, the fluorescence obtained by irradiating the excitation light that excites the fluorescent molecule from the side opposite to the solid surface of the substrate is measured from the side opposite to the solid surface of the substrate. As the substrate used, it is preferable to use a material that transmits the excitation light that excites the fluorescent molecule and the fluorescence obtained by irradiating the excitation light.
次に、前記ターゲットと前記捕捉分子とが結合している基板の固相面を、前記吸光物質を含む溶液に、前記基板の固相面が接触するように保持する。
Next, the solid-phase surface of the substrate to which the target and the capture molecule are bonded is held so that the solid-phase surface of the substrate is in contact with the solution containing the absorbent substance.
例えば、サンプル溶液中のターゲットを蛍光分子で修飾し、前記ターゲットに特異的に結合する捕捉分子が基板の固相面に固定化されている場合は、サンプル溶液中の蛍光分子で修飾されたターゲットは、前記基板の固相面に固定化された前記捕捉分子に結合する。この蛍光分子で修飾されたターゲットと、前記捕捉分子とが結合した基板の固相面を、前記吸光物質を含む溶液に、前記基板の固相面が接触するように保持する。
For example, when the target in the sample solution is modified with a fluorescent molecule and the capture molecule that specifically binds to the target is immobilized on the solid phase surface of the substrate, the target modified with the fluorescent molecule in the sample solution. Bonds to the trapping molecule immobilized on the solid surface of the substrate. The solid phase surface of the substrate to which the target modified with the fluorescent molecule and the capture molecule are bonded is held so that the solid phase surface of the substrate is in contact with the solution containing the absorbent substance.
例えば、サンプル溶液中のターゲットに特異的に結合する捕捉分子を蛍光分子で修飾し、前記ターゲットが基板の固相面に固定化されている場合は、サンプル溶液中の蛍光分子で修飾された前記捕捉分子は、基板の固相面に固定化された前記ターゲットに結合する。この蛍光分子で修飾された捕捉分子と、前記ターゲットとが結合した基板の固相面を、前記吸光物質を含む溶液に、前記基板の固相面が接触するように保持する。
For example, the capture molecule that specifically binds to the target in the sample solution is modified with a fluorescent molecule, and when the target is immobilized on the solid phase surface of the substrate, the target is modified with the fluorescent molecule in the sample solution. The trapping molecule binds to the target immobilized on the solid surface of the substrate. The solid-phase surface of the substrate to which the capture molecule modified with the fluorescent molecule and the target are bonded is held so that the solid-phase surface of the substrate is in contact with the solution containing the absorbent substance.
基板を、前記吸光物質を含む溶液に、前記基板の固相面が接触するように保持する方法としては、前記溶液が保持された容器に、前記基板の固相面が前記溶液と接触するように基板を配置する方法、前記基板の固相面が容器内部になるように基板と一体的に作製した容器に、前記溶液を基板の固相面が前記溶液に接触するように注入する方法等が挙げられる。前記基板の固相面が容器内部になるように一体的に作製した容器に、前記溶液を、前記基板の固相面が前記溶液に接触するように注入する場合は、前記溶液を注入する容器は、前記溶液を注入する注入口と、注入後に注入口をシールできる構造にしておくことが好ましい。なお、吸光物質は、予め容器に添加しておき、容器にターゲット又は捕捉分子を含むサンプル溶液を注入したときに、前記吸光物質が前記サンプル溶液に添加されるようにしておいてもよく、ターゲット又は捕捉分子を含むサンプル溶液に前記吸光物質を添加してから容器に注入してもよい。
As a method of holding the substrate in contact with the solution containing the light-absorbing substance so that the solid-phase surface of the substrate is in contact with the solution, the solid-phase surface of the substrate is brought into contact with the container in which the solution is held. A method of arranging the substrate in a container, a method of injecting the solution into a container integrally prepared with the substrate so that the solid phase surface of the substrate is inside the container, and the like so that the solid phase surface of the substrate comes into contact with the solution. Can be mentioned. When the solution is injected into a container integrally prepared so that the solid phase surface of the substrate is inside the container so that the solid phase surface of the substrate is in contact with the solution, the container into which the solution is injected. It is preferable to have a structure that can seal the injection port into which the solution is injected and the injection port after injection. The absorbent substance may be added to the container in advance so that the absorbent substance is added to the sample solution when the sample solution containing the target or the capture molecule is injected into the container. Alternatively, the absorbent substance may be added to the sample solution containing the trapping molecule and then injected into the container.
吸光物質をサンプル溶液に添加するタイミングとしては、蛍光分子に励起光を照射する前であればいかなる段階でもよく、例えば、サンプル溶液を調製する段階でも、ターゲットと捕捉分子とが基板の固相面上で結合する前の段階でもよく、また、ターゲットと捕捉分子とが基板の固相面上で結合した後の段階であってもよい。
The timing of adding the absorbent substance to the sample solution may be any stage as long as it is before the fluorescent molecule is irradiated with the excitation light. For example, even at the stage of preparing the sample solution, the target and the capture molecule are on the solid surface of the substrate. It may be a stage before the bond on the surface, or it may be a stage after the target and the trapping molecule are bonded on the solid surface surface of the substrate.
ターゲット又は捕捉分子を蛍光分子で修飾し、前記ターゲットと前記捕捉分子とが結合している基板の固相面を、前記蛍光分子を励起する励起光を吸収する吸光物質を含む溶液に、前記基板の固相面が接触するように保持することにより、溶液中に前記蛍光分子を励起する励起光を照射したときに、溶液中に含まれる吸光物質が溶液を透過しようとする励起光を吸収し、励起光の透過を抑制することができる。励起光の溶液への透過を抑制することにより、溶液の中で遊離している蛍光分子の励起を抑制することができ、溶液からの蛍光の発生を抑制し、背景光を低減することができる。また、吸光物質として、蛍光分子から発せられる蛍光を吸収する物質を用いた場合は、溶液中の前記蛍光物質が励起光により励起されて蛍光が発せられても、前記吸光物質が、溶液中から発せられる蛍光を吸収することにより、溶液からの蛍光の発生を抑制し、背景光を低減することができる。
The substrate or the capture molecule is modified with a fluorescent molecule, and the solid phase surface of the substrate to which the target and the capture molecule are bonded is placed in a solution containing an absorbent substance that absorbs the excitation light that excites the fluorescent molecule. By holding the solid phase surface of the solution in contact with each other, when the solution is irradiated with the excitation light that excites the fluorescent molecule, the absorbent substance contained in the solution absorbs the excitation light that tries to pass through the solution. , Transmission of excitation light can be suppressed. By suppressing the transmission of the excitation light into the solution, the excitation of the fluorescent molecules free in the solution can be suppressed, the generation of fluorescence from the solution can be suppressed, and the background light can be reduced. .. When a substance that absorbs fluorescence emitted from a fluorescent molecule is used as the light-absorbing substance, even if the fluorescent substance in the solution is excited by excitation light to emit fluorescence, the light-absorbing substance is released from the solution. By absorbing the emitted fluorescence, it is possible to suppress the generation of fluorescence from the solution and reduce the background light.
また、ターゲットを含む溶液の調製時に、生体や微生物等を含む検体から抽出したサンプル溶液中に含まれる検体由来のタンパク質や糖類等の残留分子が、ターゲットを含む溶液に含まれる場合がある。その場合であっても、吸光物質は、該残留分子の励起又は蛍光の発生を抑制することができるため、本来洗浄を必要としないターゲット検出用デバイスに適用する場合でも、該残留物質から発生する蛍光による背景光を低減することができ、検出感度をより向上させることができる。
In addition, when preparing a solution containing a target, residual molecules such as proteins and sugars derived from the sample contained in the sample solution extracted from the sample containing a living body or a microorganism may be contained in the solution containing the target. Even in that case, since the light-absorbing substance can suppress the generation of excitation or fluorescence of the residual molecule, it is generated from the residual substance even when applied to a target detection device that originally does not require cleaning. The background light due to fluorescence can be reduced, and the detection sensitivity can be further improved.
吸光物質としては、蛍光分子を励起する励起光、又は蛍光分子から発せられる蛍光の波長の光を吸収する光学特性を有するものであれば特に制限はなく、本発明に用いられる蛍光分子が発生する蛍光の波長に応じ適宜選択されるが、塗料、インク、化粧品、食品などの着色に用いられる顔料等が用いられる。顔料は金や銀などの金属、酸化鉄などの酸化物、窒化物、有機ポリマーなど様々な粒子があり、物質材料そのものの吸光特性や着色粒子、表面着色による吸光の光学特性から選択して吸光物質として用いることができる。また、金属ナノ粒子は粒子表面の電子と光の相互作用によって特定の波長で表面プラズモン共鳴が生じ、光の強い減光が生じるため、吸光物質として用いることができる。
The absorbent material is not particularly limited as long as it has an excitation light that excites a fluorescent molecule or an optical property that absorbs light having a wavelength of fluorescence emitted from the fluorescent molecule, and the fluorescent molecule used in the present invention is generated. Although it is appropriately selected according to the wavelength of fluorescence, pigments and the like used for coloring paints, inks, cosmetics, foods and the like are used. Pigments include various particles such as metals such as gold and silver, oxides such as iron oxide, nitrides, and organic polymers. Absorption is selected from the absorption characteristics of the material itself, colored particles, and the optical characteristics of absorption due to surface coloring. It can be used as a substance. In addition, metal nanoparticles can be used as an absorbent because surface plasmon resonance occurs at a specific wavelength due to the interaction between electrons and light on the particle surface and strong dimming of light occurs.
本発明に用いられる吸光物質の具体例としては、例えば、蛍光分子としてＣｙ３（登録商標）を用いる場合は、酸化鉄（Ｆｅ２Ｏ３、Ｆｅ２Ｏ４）、金ナノ粒子、銀ナノ粒子、黒色着色シリカ粒子、スチレンとアクリル酸共重合体などのポリマー黒色着色粒子等が挙げられる。なお、吸光物質は乾燥状態であっても、溶液状態であってもよい。
Specific examples of the absorbent substance used in the present invention include, for example, when Cy3 (registered trademark) is used as a fluorescent molecule, iron oxide (Fe 2 O 3 , Fe 2 O 4 ), gold nanoparticles, silver nanoparticles, and the like. Examples thereof include black colored silica particles, polymer black colored particles such as a styrene and acrylic acid copolymer, and the like. The absorbent material may be in a dry state or in a solution state.
吸光物質が、励起光を吸収する物質である場合は、固相を透過し、吸光物質を含む溶液に入ってくる励起光の散乱が小さくなる吸光物質が好ましい。これは、吸光物質を含む溶液に入ってくる励起光の散乱が小さくなる吸光物質であれば、溶液中を透過する光が吸光物質で生じる散乱により光路長が伸び、溶液中の蛍光分子を余計に励起し蛍光が増加する現象をより抑制することができるためである。
When the absorbent substance is a substance that absorbs the excitation light, an absorbent substance that penetrates the solid phase and reduces the scattering of the excitation light that enters the solution containing the absorbent substance is preferable. This is because if the scattering of the excitation light entering the solution containing the absorbent substance is small, the light path length is extended due to the scattering of the light transmitted through the solution due to the scattering substance, and the fluorescent molecules in the solution are added. This is because it is possible to further suppress the phenomenon of being excited by the substance and increasing the fluorescence.
この現象は、入射光と物質を透過した透過光の比の対数を吸光度として表し、濃度や光路長と比例関係にあることを定式化した法則であるＬａｍｂｅｒｔ－Ｂｅｅｒ則に対して散乱の影響を加味したＭｏｄｉｆｉｅｄ Ｌａｍｂｅｒｔ－Ｂｅｅｒ則で定式化されている現象に類似し、散乱物質が存在するときに該物質の濃度や光軸方向の直線的な光路長に比例した吸光度の増加が得られない現象である。光の物質による散乱現象は粒径により異なり、波長より十分大きい物質の場合は、幾何光学近似、波長程度の粒径の場合は、ミー散乱、波長より十分小さい粒径の場合は、レイリー散乱で表される。本発明においては、吸光物質は、溶液中に添加され分散されるため、ミー散乱又はレイリー散乱が起こると考えられる。ミー散乱の全散乱強度は粒径により異なり、粒径の２乗から６乗に比例して大きくなることが知られている。また、レイリー散乱の全散乱強度は粒径の６乗に比例して大きくなることが知られている。上記の理由により、本発明においては、吸光物質の粒径は、小さい方が好ましい。
This phenomenon expresses the logarithm of the ratio of the incident light and the transmitted light transmitted through the substance as the absorbance, and has the effect of scattering on the Lambert-Beer law, which is a law that formulates a proportional relationship with the concentration and the optical path length. Similar to the phenomenon formulated by the added Modified Lambert-Beer law, a phenomenon in which when a scattering substance is present, the increase in absorbance in proportion to the concentration of the substance and the linear optical path length in the optical axis direction cannot be obtained. Is. The scattering phenomenon of light by a substance differs depending on the particle size. For a substance sufficiently larger than the wavelength, it is a geometrical optical approximation, for a particle size of about the wavelength, Mie scattering, and for a particle size sufficiently smaller than the wavelength, Rayleigh scattering. expressed. In the present invention, since the absorbent substance is added and dispersed in the solution, it is considered that Mie scattering or Rayleigh scattering occurs. It is known that the total scattering intensity of Mie scattering varies depending on the particle size and increases in proportion to the square to the sixth power of the particle size. It is also known that the total scattering intensity of Rayleigh scattering increases in proportion to the sixth power of the particle size. For the above reasons, in the present invention, it is preferable that the particle size of the absorbent substance is small.
吸光物質が、蛍光を吸収する物質である場合は、溶液と固相面との接触面近くの蛍光分子からの蛍光が溶液内部の蛍光分子からの蛍光よりも早く、基板の固相面とは反対側に設置された検出器に到達するため、溶液と固相との接触面近くで発生した蛍光が、溶液側で散乱されて検出器側に到達せずに、吸光物質で吸収された方が好ましいため、蛍光の散乱が小さくなる吸光物質が好ましい。
When the absorbent substance is a substance that absorbs fluorescence, the fluorescence from the fluorescent molecule near the contact surface between the solution and the solid phase surface is faster than the fluorescence from the fluorescent molecule inside the solution, and the solid phase surface of the substrate is Those in which the fluorescence generated near the contact surface between the solution and the solid phase is scattered on the solution side and is absorbed by the absorbent substance without reaching the detector side in order to reach the detector installed on the opposite side. Therefore, an absorbent substance that reduces the scattering of fluorescence is preferable.
また、吸光物質は溶液中に添加するため、蛍光の計測中に安定的に分散し、溶液中でムラなどが生じないことが望ましい。粒子の分散安定性は、目視観察、透過光強度変化の測定、散乱光強度変化の測定など様々な測定方法により測定される。例えば、物質を遠心分離機により遠心沈降させるための遠心分離条件のパラメータから沈殿のし難さを推定し、分散安定性を推定することができる。ポリマー粒子では粒径８００ｎｍ未満で遠心分離に必要なパラメータが１０，０００×ｇ、２０分となり沈殿し難く、分散性に優れており好ましい。吸光物質がシリカ粒子の場合は粒径が２００ｎｍ以下、酸化鉄粒子の場合は粒径が１００ｎｍ以下、金ナノ粒子の場合は粒径が１５ｎｍ以下の粒子が分散性に優れており好ましい。
In addition, since the absorbent substance is added to the solution, it is desirable that it is stably dispersed during the measurement of fluorescence and that unevenness does not occur in the solution. The dispersion stability of particles is measured by various measuring methods such as visual observation, measurement of change in transmitted light intensity, and measurement of change in scattered light intensity. For example, the difficulty of precipitation can be estimated from the parameters of the centrifugation conditions for centrifuging the substance by a centrifuge, and the dispersion stability can be estimated. For polymer particles, the particle size is less than 800 nm, the parameter required for centrifugation is 10,000 × g, which is 20 minutes, and it is difficult to precipitate, and it is excellent in dispersibility, which is preferable. When the absorptive substance is silica particles, the particle size is 200 nm or less, when it is iron oxide particles, the particle size is 100 nm or less, and when it is gold nanoparticles, particles having a particle size of 15 nm or less are excellent in dispersibility and are preferable.
また、本発明においては、吸光物質は、溶液に添加し分散させるために親水性であることが好ましい。吸光物質が、疎水性である場合は、表面の親水性への改質や、カルボキシル基、スルホン基などの表面官能基の導入、酸化物による被覆、ＰＥＧやＰＥＯ、デキストランなどの親水性ポリマーの化学修飾などにより親水化することが好ましい。
Further, in the present invention, the absorbent substance is preferably hydrophilic in order to be added to and dispersed in the solution. If the absorptive substance is hydrophobic, it can be modified to be hydrophilic on the surface, introduced with surface functional groups such as carboxyl groups and sulfon groups, coated with oxides, and hydrophilic polymers such as PEG, PEO and dextran. It is preferable to make it hydrophilic by chemical modification or the like.
ターゲットと捕捉分子を結合させる前に、溶液に吸光物質を添加する場合は、添加する吸光物質がターゲットや捕捉分子を吸着しないことが好ましい。例えば、前記ＰＥＧなどの親水性ポリマーを、吸光物質の粒子の表面に修飾することにより、吸光物質が、ターゲットや捕捉分子に非特異的に吸着することを抑制することができる。また、吸光物質を含む溶液に、生体分子の非特異吸着を抑えるＢＳＡ等のブロッキング剤を添加することにより、吸光物質がターゲットや捕捉分子へ非特異的に吸着することを抑制することができる。
When an absorbent substance is added to the solution before the target and the capture molecule are bound, it is preferable that the added absorbance substance does not adsorb the target or the capture molecule. For example, by modifying the surface of the particles of the light-absorbing substance with the hydrophilic polymer such as PEG, it is possible to suppress the non-specific adsorption of the light-absorbing substance on the target or the trapping molecule. Further, by adding a blocking agent such as BSA that suppresses non-specific adsorption of biomolecules to a solution containing an absorbent substance, it is possible to suppress non-specific adsorption of the absorbent substance on a target or a trapping molecule.
次に、前記基板の固相面とは反対側から蛍光分子を励起する励起光を照射し、得られる蛍光を、前記基板の固相面とは反対側から計測する。
Next, the excitation light that excites the fluorescent molecule is irradiated from the side opposite to the solid surface surface of the substrate, and the obtained fluorescence is measured from the side opposite to the solid surface surface of the substrate.
前記基板の固相面とは反対側から蛍光分子を励起する励起光を照射し、得らえる蛍光を計測する方法としては、蛍光分子から発せられる蛍光を計測することができれば特に制限はないが、例えば、カメラを用い、カメラの検出素子上に、蛍光分子から発生する蛍光画像を結合させることによって、計測する方法等が挙げられる。
The method of irradiating the excitation light that excites the fluorescent molecule from the side opposite to the solid phase surface of the substrate and measuring the obtained fluorescence is not particularly limited as long as the fluorescence emitted from the fluorescent molecule can be measured. For example, a method of measuring by using a camera and binding a fluorescent image generated from a fluorescent molecule onto a detection element of the camera can be mentioned.
励起光源としては、例えば、単波長のレーザー光又はそのエキスパンド光を射出するレーザー光源、ＬＥＤ（Ｌｉｇｈｔ Ｅｍｉｔｔｉｎｇ Ｄｉｏｄｅ：発光ダイオード）、白色光を放出するランプ、ＬＥＤと波長フィルタとの組合せからなる光源等を用いることができる。
Examples of the excitation light source include a laser light source that emits a single-wavelength laser beam or its expanded light, an LED (Light Emitting Diode), a lamp that emits white light, a light source composed of a combination of an LED and a wavelength filter, and the like. Can be used.
計測に使用するカメラはカラー及びモノクロＣＣＤ、ＣＭＯＳカメラを始め、高感度を特徴とするＥＭ－ＣＣＤやデジタルＣＭＯＳ等を用いることができる。またスポットと１対１で配置した単一検出器であるフォトダイオードなどの組み合わせでもよい。
The camera used for measurement can be a color or monochrome CCD, a CMOS camera, an EM-CCD characterized by high sensitivity, a digital CMOS, or the like. Further, a combination such as a photodiode which is a single detector arranged one-to-one with the spot may be used.
本発明のターゲット計測方法により得られる蛍光画像は、同一スポットのターゲットと捕捉分子の結合前後の画像を取得することができる。そのため、固相間、スポット間の光量のばらつきの影響を受けない。また、結合前後の蛍光画像から蛍光変化量を演算し、結合した分子数を算出することができる。蛍光変化量の演算は、スポット全体の平均光量を使用してもよいし、スポット画像の各ピクセルの蛍光変化量を使用してもよい。
As the fluorescent image obtained by the target measurement method of the present invention, it is possible to acquire images before and after the binding between the target at the same spot and the captured molecule. Therefore, it is not affected by variations in the amount of light between solid phases and spots. In addition, the amount of change in fluorescence can be calculated from the fluorescence images before and after binding, and the number of bound molecules can be calculated. The calculation of the fluorescence change amount may use the average light amount of the entire spot, or may use the fluorescence change amount of each pixel of the spot image.
次に、本発明のターゲット計測用デバイスについて説明する。本発明のターゲット計測用デバイスは、本発明のターゲット計測方法に用いることができる。
本発明のターゲット計測用デバイスは、サンプルに含まれるターゲットを計測する際に用いられるターゲット計測用デバイスであって、前記ターゲットと前記捕捉分子とのいずれか一方が固相面に固定化される基板と、前記ターゲットと前記捕捉分子との結合物を修飾する蛍光分子を励起する励起光、又は前記蛍光分子から発せられる蛍光を吸収する吸光物質が添加された容器であって、前記ターゲット及び前記捕捉分子のいずれか他方と、前記吸光物質と、を含む溶液を、前記基板の前記固相面に接触させた状態で保持可能な容器と、を備えたターゲット計測用デバイスである。 Next, the target measurement device of the present invention will be described. The target measurement device of the present invention can be used in the target measurement method of the present invention.
The target measurement device of the present invention is a target measurement device used when measuring a target contained in a sample, and is a substrate in which either the target or the capture molecule is immobilized on a solid phase surface. And the container to which the excitation light that excites the fluorescent molecule that modifies the bond between the target and the capture molecule, or the absorbent substance that absorbs the fluorescence emitted from the fluorescent molecule is added, and the target and the capture. A device for target measurement comprising one of the molecules and a container capable of holding a solution containing the absorbent substance in contact with the solid phase surface of the substrate.
本発明のターゲット計測用デバイスは、サンプルに含まれるターゲットを計測する際に用いられるターゲット計測用デバイスであって、前記ターゲットと前記捕捉分子とのいずれか一方が固相面に固定化される基板と、前記ターゲットと前記捕捉分子との結合物を修飾する蛍光分子を励起する励起光、又は前記蛍光分子から発せられる蛍光を吸収する吸光物質が添加された容器であって、前記ターゲット及び前記捕捉分子のいずれか他方と、前記吸光物質と、を含む溶液を、前記基板の前記固相面に接触させた状態で保持可能な容器と、を備えたターゲット計測用デバイスである。 Next, the target measurement device of the present invention will be described. The target measurement device of the present invention can be used in the target measurement method of the present invention.
The target measurement device of the present invention is a target measurement device used when measuring a target contained in a sample, and is a substrate in which either the target or the capture molecule is immobilized on a solid phase surface. And the container to which the excitation light that excites the fluorescent molecule that modifies the bond between the target and the capture molecule, or the absorbent substance that absorbs the fluorescence emitted from the fluorescent molecule is added, and the target and the capture. A device for target measurement comprising one of the molecules and a container capable of holding a solution containing the absorbent substance in contact with the solid phase surface of the substrate.
本発明のターゲット計測用デバイスとしては、基板の固相面に捕捉分子が固定化されており、容器に、蛍光分子で修飾されたターゲットを含む溶液が供給される、ターゲット計測用デバイス；基板の固相面にターゲットが固定化されており、容器に、蛍光分子で修飾された捕捉分子を含む溶液が供給されるターゲット計測用デバイス；基板の固相面に蛍光分子で修飾されている捕捉分子が固定化されており、容器に、前記ターゲットを含む溶液が供給されるターゲット計測用デバイス；基板の固相面に、ターゲット及び捕捉分子のいずれか一方が固定化されており、容器に、前記ターゲット及び前記捕捉分子のいずれか他方と、前記ターゲットと前記捕捉分子との結合体に結合する蛍光分子とを含む溶液が供給されるターゲット計測用デバイス等が挙げられる。ターゲット及び捕捉分子としては、前述したものが挙げられる。
The target measurement device of the present invention is a target measurement device in which a trapping molecule is immobilized on a solid phase surface of a substrate and a solution containing a target modified with a fluorescent molecule is supplied to a container; Target measurement device in which a target is immobilized on a solid surface and a solution containing a capture molecule modified with a fluorescent molecule is supplied to a container; a capture molecule modified with a fluorescent molecule on the solid surface of a substrate. Is immobilized and a target measuring device is supplied with a solution containing the target in a container; one of a target and a capture molecule is immobilized on the solid phase surface of the substrate, and the container is equipped with the above. Examples thereof include a target measurement device to which a solution containing any one of the target and the capture molecule and a fluorescent molecule that binds to a conjugate of the target and the capture molecule is supplied. Examples of the target and the capture molecule include those described above.
図４は、本発明のターゲット計測用デバイスの構成の一例を示す図である。図４では、固相として、ＤＮＡプローブが固定化されたＤＮＡマイクロアレイの例を示している。
FIG. 4 is a diagram showing an example of the configuration of the target measurement device of the present invention. FIG. 4 shows an example of a DNA microarray on which a DNA probe is immobilized as a solid phase.
本発明のターゲット計測用デバイスは、基板の固相面には、ターゲット又は捕捉分子が固定化されている。図４では、ターゲットとなる特定の核酸配列の相補配列を有するＤＮＡプローブ１が、基板であるＤＮＡマイクロアレイ５に固定化されている。本実施形態のターゲット計測用デバイスは、ターゲット３を蛍光分子４で修飾し、蛍光分子４で修飾されたターゲット３と、前記蛍光分子４を励起する励起光３３又は、前記蛍光分子４から発せられる蛍光３４を吸収する吸光物質と、を含む吸光物質添加ターゲット溶液３５を保持することが可能な容器３２を、前記吸光物質添加ターゲット溶液３５が、前記ＤＮＡプローブ１が固定化されているＤＮＡマイクロアレイ５の固相面に接触させた状態で保持している。
In the target measurement device of the present invention, a target or a trapping molecule is immobilized on the solid phase surface of the substrate. In FIG. 4, a DNA probe 1 having a complementary sequence of a specific target nucleic acid sequence is immobilized on a DNA microarray 5 which is a substrate. The target measurement device of the present embodiment is emitted from the target 3 in which the target 3 is modified with the fluorescent molecule 4 and the fluorescent molecule 4 is modified, and the excitation light 33 for exciting the fluorescent molecule 4 or the fluorescent molecule 4. A container 32 capable of holding an absorbent target solution 35 containing an absorbent substance that absorbs fluorescence 34, and a DNA microarray 5 in which the absorbent substance-added target solution 35 is immobilized with the DNA probe 1. It is held in contact with the solid phase surface of.
次に、ターゲット計測用デバイスによりターゲット３を検出する原理及び操作手順について、図４に示したターゲット計測用デバイスを基に説明する。図５は、図４に示したターゲット計測用デバイスを用いてターゲット３を検出する操作手順を示すフローチャートである。
Next, the principle and operation procedure for detecting the target 3 by the target measurement device will be described based on the target measurement device shown in FIG. FIG. 5 is a flowchart showing an operation procedure for detecting the target 3 using the target measurement device shown in FIG.
まず、基板に蛍光分子４で修飾されたＤＮＡプローブ１をＤＮＡスポット３０に固定化する（ステップＳ１）。次に、サンプル中のターゲット３を蛍光分子４で修飾し、ターゲット溶液を調製する（ステップＳ２）。ターゲット溶液を調製する際に、特定の核酸配列を有するターゲット３の増幅を行ってもよい。サンプル中にターゲット３が存在するか否かを確認するタイミングとしては、増幅終了後に限定されず、増幅中であってもよい。ターゲット３の増幅を行う場合は、ターゲット３の蛍光分子４での修飾は、増幅が確認されてから行い、増幅が確認された場合にのみ、後述するステップＳ３に進むようにしてもよい。なお、ターゲット３の存在を確認する方法としては、電気泳動、抗原抗体反応、質量分析、リアルタイムＰＣＲ等を適宜利用することができる。
First, the DNA probe 1 modified with the fluorescent molecule 4 is immobilized on the DNA spot 30 (step S1). Next, the target 3 in the sample is modified with the fluorescent molecule 4 to prepare a target solution (step S2). When preparing the target solution, amplification of the target 3 having a specific nucleic acid sequence may be performed. The timing for confirming whether or not the target 3 is present in the sample is not limited to that after the completion of amplification, and may be during amplification. When the target 3 is amplified, the modification of the target 3 with the fluorescent molecule 4 may be performed after the amplification is confirmed, and the process may proceed to step S3 described later only when the amplification is confirmed. As a method for confirming the presence of the target 3, electrophoresis, antigen-antibody reaction, mass spectrometry, real-time PCR and the like can be appropriately used.
次に、調製したターゲット溶液を、吸光物質が添加された容器であって、ＤＮＡプローブ１が固定化されているＤＮＡマイクロアレイが容器内部に配置されている容器３２に供給し、ターゲット溶液を、ＤＮＡマイクロアレイ５の固相面に接触させる（ステップＳ３）。容器３２に添加されている吸光物質は、ターゲット溶液を容器３２に供給する際に、ターゲット溶液に添加され、吸光物質添加ターゲット溶液３５となる。
Next, the prepared target solution is supplied to the container 32 to which the DNA microarray on which the DNA probe 1 is immobilized is arranged in the container to which the light-absorbing substance is added, and the target solution is supplied to the DNA. It is brought into contact with the solid surface of the microarray 5 (step S3). The absorbent substance added to the container 32 is added to the target solution when the target solution is supplied to the container 32, and becomes the absorbent substance-added target solution 35.
吸光物質添加ターゲット溶液３５を、ＤＮＡプローブ１が固定化されているＤＮＡマイクロアレイ５の固相面に接触させた後に、蛍光分子４で修飾されたターゲット３と、ＤＮＡマイクロアレイ５に固定化されたＤＮＡプローブ１とをハイブリダイズ反応させる（ステップＳ４）。このハイブリダイズ反応により、ターゲット３がＤＮＡプローブ１と結合し、ＤＮＡプローブ１が固定化されているＤＮＡスポット３０にターゲット３を修飾している蛍光分子４が捕捉される。
After contacting the target solution 35 to which the absorbent substance is added with the solid phase surface of the DNA microarray 5 on which the DNA probe 1 is immobilized, the target 3 modified with the fluorescent molecule 4 and the DNA immobilized on the DNA microarray 5 Hybridization reaction with probe 1 (step S4). By this hybridization reaction, the target 3 binds to the DNA probe 1, and the fluorescent molecule 4 modifying the target 3 is captured in the DNA spot 30 on which the DNA probe 1 is immobilized.
ハイブリダイズ反応後、蛍光分子４を励起する励起光３３を、ＤＮＡマイクロアレイ５の固相面とは反対側から照射する（ステップＳ５）。
After the hybridization reaction, the excitation light 33 that excites the fluorescent molecule 4 is irradiated from the side opposite to the solid phase surface of the DNA microarray 5 (step S5).
次に、ＤＮＡマイクロアレイ５に固定化されたＤＮＡプローブ１に結合したターゲット３を修飾する蛍光分子４から発生する蛍光３４を、ＤＮＡマイクロアレイ５の固相面とは反対側から検出する（ステップＳ６）。例えば、蛍光分子４から発生する蛍光画像を蛍光読取装置４０で取得する。次に、取得された蛍光画像から、蛍光量を算出する（ステップＳ７）。
Next, the fluorescence 34 generated from the fluorescent molecule 4 that modifies the target 3 bound to the DNA probe 1 immobilized on the DNA microarray 5 is detected from the side opposite to the solid phase surface of the DNA microarray 5 (step S6). .. For example, the fluorescence image generated from the fluorescence molecule 4 is acquired by the fluorescence reader 40. Next, the amount of fluorescence is calculated from the acquired fluorescence image (step S7).
蛍光分子４を励起する励起光３３を、基板であるＤＮＡマイクロアレイ５の固相面とは反対側から照射すると、ターゲット３を修飾している蛍光分子４から蛍光が放射される。
When the excitation light 33 that excites the fluorescent molecule 4 is irradiated from the side opposite to the solid phase surface of the DNA microarray 5 that is the substrate, fluorescence is emitted from the fluorescent molecule 4 that modifies the target 3.
吸光物質が蛍光分子４を励起する励起光３３を吸収する物質である場合は、蛍光分子４を励起するための励起光３３は、吸光物質添加ターゲット溶液３５に照射されるが、吸光物質添加ターゲット溶液３５には、蛍光分子４を励起する励起光３３を吸収する吸光物質を含んでいるため、前記溶液３５中で遊離している、ＤＮＡプローブ１とハイブリダイズしていない未反応の蛍光分子４で修飾されたターゲット３の蛍光分子４の励起を抑制することができる。また、吸光物質が蛍光分子４から発せられる蛍光３４を吸収する物質である場合は、蛍光分子４を励起するための励起光３３は、吸光物質添加ターゲット溶液３５に照射されるが、吸光物質添加ターゲット溶液３５には、蛍光分子４から発せられる蛍光３４を吸収する吸光物質を含んでいるため、前記溶液３５中で遊離している、ＤＮＡプローブ１とハイブリダイズしていない未反応の蛍光分子４で修飾されたターゲット３の蛍光分子４からの蛍光の発生を抑制することができる。これにより、前記溶液３５が蛍光を呈することを抑制できるため、固相を洗浄することなく、ターゲット３を含む溶液存在下で、固相に結合したターゲット３から発生する蛍光を計測することができる。また、ハイブリダイズ反応中のリアルタイム測定も可能となる。
When the light-absorbing substance is a substance that absorbs the excitation light 33 that excites the fluorescent molecule 4, the excitation light 33 for exciting the fluorescent molecule 4 is irradiated to the light-absorbing substance-added target solution 35, but the light-absorbing substance-added target Since the solution 35 contains an absorbent substance that absorbs the excitation light 33 that excites the fluorescent molecule 4, the unreacted fluorescent molecule 4 that is free in the solution 35 and does not hybridize with the DNA probe 1 is released. It is possible to suppress the excitation of the fluorescent molecule 4 of the target 3 modified with. When the light-absorbing substance is a substance that absorbs the fluorescence 34 emitted from the fluorescent molecule 4, the excitation light 33 for exciting the fluorescent molecule 4 is irradiated to the target solution 35 to which the light-absorbing substance is added, but the light-absorbing substance is added. Since the target solution 35 contains an absorbent substance that absorbs the fluorescence 34 emitted from the fluorescent molecule 4, the unreacted fluorescent molecule 4 that is free in the solution 35 and does not hybridize with the DNA probe 1 It is possible to suppress the generation of fluorescence from the fluorescent molecule 4 of the target 3 modified with. As a result, it is possible to suppress the fluorescence of the solution 35, so that the fluorescence generated from the target 3 bonded to the solid phase can be measured in the presence of the solution containing the target 3 without washing the solid phase. .. In addition, real-time measurement during the hybridization reaction becomes possible.
また、本発明のターゲット計測方法により、ハイブリダイズ反応前後の蛍光分子４の蛍光変化量から、ハイブリダイズ反応したターゲット３の分子数を算出することができる。例えば、既知の分子数を有するターゲット３の標準液を用いてハイブリダイズ反応を行い、反応前後の蛍光分子４の蛍光変化量を測定して、分子数と蛍光変化量の関係を示した検量線を予め作成しておく。この検量線と、サンプルを用いたハイブリダイズ反応前後の蛍光分子４の蛍光変化量とから、ハイブリダイズ反応したターゲット３の分子数を算出することができる。ハイブリダイズ反応前後の蛍光分子４の蛍光変化量を測定するためにはステップＳ２またはステップＳ３において、吸光物質を溶液に添加しておく。
Further, by the target measurement method of the present invention, the number of molecules of the hybridized target 3 can be calculated from the amount of change in fluorescence of the fluorescent molecule 4 before and after the hybridization reaction. For example, a hybrid reaction is carried out using a standard solution of Target 3 having a known number of molecules, the amount of change in fluorescence of fluorescent molecule 4 before and after the reaction is measured, and a calibration curve showing the relationship between the number of molecules and the amount of change in fluorescence. Is created in advance. From this calibration curve and the amount of change in fluorescence of the fluorescent molecule 4 before and after the hybridization reaction using the sample, the number of molecules of the hybridized target 3 can be calculated. In order to measure the amount of change in fluorescence of the fluorescent molecule 4 before and after the hybridization reaction, an absorbent substance is added to the solution in step S2 or step S3.
次に、本発明のターゲット計測方法にかかるターゲット計測用デバイスの製造方法について説明する。
Next, a method of manufacturing a target measurement device according to the target measurement method of the present invention will be described.
（１）溶液調製
まず、ターゲット又はターゲットに特異的に結合する捕捉分子を含む溶液を調製し、ターゲット又は捕捉分子の濃度を調整する。例えば、ターゲットが特定の核酸配列を有するＤＮＡであって、捕捉分子が、ターゲットの特定の核酸配列の相補的な配列を有するＤＮＡプローブである場合は、ＤＮＡプローブ溶液を調製し、ＤＮＡプローブ溶液の濃度を調整する。 (1) Solution preparation First, a solution containing a target or a capture molecule that specifically binds to the target is prepared, and the concentration of the target or the capture molecule is adjusted. For example, if the target is DNA having a specific nucleic acid sequence and the capture molecule is a DNA probe having a complementary sequence to the specific nucleic acid sequence of the target, a DNA probe solution is prepared and the DNA probe solution is prepared. Adjust the concentration.
まず、ターゲット又はターゲットに特異的に結合する捕捉分子を含む溶液を調製し、ターゲット又は捕捉分子の濃度を調整する。例えば、ターゲットが特定の核酸配列を有するＤＮＡであって、捕捉分子が、ターゲットの特定の核酸配列の相補的な配列を有するＤＮＡプローブである場合は、ＤＮＡプローブ溶液を調製し、ＤＮＡプローブ溶液の濃度を調整する。 (1) Solution preparation First, a solution containing a target or a capture molecule that specifically binds to the target is prepared, and the concentration of the target or the capture molecule is adjusted. For example, if the target is DNA having a specific nucleic acid sequence and the capture molecule is a DNA probe having a complementary sequence to the specific nucleic acid sequence of the target, a DNA probe solution is prepared and the DNA probe solution is prepared. Adjust the concentration.
（２）固相面への固定
ターゲット又は捕捉分子を基板の固相面に固定化する。例えば、ＤＮＡプローブを固相面に固定化する場合は、ＤＮＡプローブ１を固相面にスポッター等を用いてスポットして、ＤＮＡプローブ１を固相面に固定化する。固相面は、ブロッキング液に液浸し、未反応の活性官能基を不活性化する。 (2) Immobilization on the solid surface The target or trapping molecule is immobilized on the solid surface of the substrate. For example, when the DNA probe is immobilized on the solid surface, the DNA probe 1 is spotted on the solid surface using a spotter or the like, and the DNA probe 1 is immobilized on the solid surface. The solid phase surface is immersed in a blocking solution to inactivate unreacted active functional groups.
ターゲット又は捕捉分子を基板の固相面に固定化する。例えば、ＤＮＡプローブを固相面に固定化する場合は、ＤＮＡプローブ１を固相面にスポッター等を用いてスポットして、ＤＮＡプローブ１を固相面に固定化する。固相面は、ブロッキング液に液浸し、未反応の活性官能基を不活性化する。 (2) Immobilization on the solid surface The target or trapping molecule is immobilized on the solid surface of the substrate. For example, when the DNA probe is immobilized on the solid surface, the DNA probe 1 is spotted on the solid surface using a spotter or the like, and the DNA probe 1 is immobilized on the solid surface. The solid phase surface is immersed in a blocking solution to inactivate unreacted active functional groups.
ターゲット又は捕捉分子をスポットする固相面上の領域は、予め規定された数を単位としたブロック毎に区分けされていてもよい。ターゲット計測用デバイスに対するターゲット溶液の添加は、ブロック毎に行われる。また、ターゲット計測用デバイスの画像取得は、ブロック毎に行われることが多い。つまり、ブロックは、画像取得領域であるということができる。
The region on the solid phase surface where the target or the trapping molecule is spotted may be divided into blocks in units of a predetermined number. The addition of the target solution to the target measurement device is performed block by block. Further, the image acquisition of the target measurement device is often performed for each block. That is, it can be said that the block is an image acquisition area.
（３）洗浄
次に、固相面を洗浄し、固定化されていない余剰のターゲット又は捕捉分子を除去し、洗浄液も除去する。以上の手順により製造された基板は、遮光、温度、湿度条件等、基板及び基板に固定化したターゲットや捕捉分子の性質に適した環境下で使用まで適宜保存される。 (3) Washing Next, the solid phase surface is washed to remove excess unimmobilized targets or trapping molecules, and the washing liquid is also removed. The substrate produced by the above procedure is appropriately stored until use in an environment suitable for the substrate and the properties of the target and the trapping molecule immobilized on the substrate, such as shading, temperature, and humidity conditions.
次に、固相面を洗浄し、固定化されていない余剰のターゲット又は捕捉分子を除去し、洗浄液も除去する。以上の手順により製造された基板は、遮光、温度、湿度条件等、基板及び基板に固定化したターゲットや捕捉分子の性質に適した環境下で使用まで適宜保存される。 (3) Washing Next, the solid phase surface is washed to remove excess unimmobilized targets or trapping molecules, and the washing liquid is also removed. The substrate produced by the above procedure is appropriately stored until use in an environment suitable for the substrate and the properties of the target and the trapping molecule immobilized on the substrate, such as shading, temperature, and humidity conditions.
（４）吸光物質を含む溶液を保持する容器への基板の配置
（３）で得られたターゲット又は捕捉分子が固定化された基板を、ターゲット又は捕捉分子が固定化されている固相面が、吸光物質を含む溶液を保持するための容器側になるように、前記容器に配置する。基板は、容器と一体化しても、基板と容器を別にして、ターゲットの計測時に基板を容器に配置できようにしてもよい。なお、基板を容器と一体化している場合は、容器に溶液を注入できる注入口と注入後に注入口をシールできる構造を有することが好ましい。 (4) Placement of the substrate on the container holding the solution containing the light-absorbing substance The substrate on which the target or capture molecule is immobilized in (3) has a solid phase surface on which the target or capture molecule is immobilized. , Place the container so that it is on the side of the container for holding the solution containing the absorbance. The substrate may be integrated with the container, or the substrate and the container may be separated so that the substrate can be arranged in the container when measuring the target. When the substrate is integrated with the container, it is preferable to have an injection port capable of injecting the solution into the container and a structure capable of sealing the injection port after the injection.
（３）で得られたターゲット又は捕捉分子が固定化された基板を、ターゲット又は捕捉分子が固定化されている固相面が、吸光物質を含む溶液を保持するための容器側になるように、前記容器に配置する。基板は、容器と一体化しても、基板と容器を別にして、ターゲットの計測時に基板を容器に配置できようにしてもよい。なお、基板を容器と一体化している場合は、容器に溶液を注入できる注入口と注入後に注入口をシールできる構造を有することが好ましい。 (4) Placement of the substrate on the container holding the solution containing the light-absorbing substance The substrate on which the target or capture molecule is immobilized in (3) has a solid phase surface on which the target or capture molecule is immobilized. , Place the container so that it is on the side of the container for holding the solution containing the absorbance. The substrate may be integrated with the container, or the substrate and the container may be separated so that the substrate can be arranged in the container when measuring the target. When the substrate is integrated with the container, it is preferable to have an injection port capable of injecting the solution into the container and a structure capable of sealing the injection port after the injection.
（５）吸光物質の容器への添加
（４）で調製した容器に吸光物質を添加する。吸光物質は、乾燥状態であっても液状であってもよい。容器に吸光物質を予め添加しておくことで、蛍光分子で修飾されたターゲット又は捕捉分子を含む溶液を容器に供給した際に、前記溶液に吸光物質を添加することができる。なお、吸光物質の容器への添加は、基板を容器に配置する前に行ってもよく、基板を容器に配置した後でもよい。吸光物質の添加を、基板を容器に配置した後に行う場合は、吸光物質は容器に設けた注入口から添加することができる。 (5) Addition of the absorbent substance to the container Add the absorbent substance to the container prepared in (4). The absorbent material may be in a dry state or in a liquid state. By adding the absorbent substance to the container in advance, the absorbent substance can be added to the solution when the solution containing the target or the capture molecule modified with the fluorescent molecule is supplied to the container. The absorption substance may be added to the container before the substrate is placed in the container, or may be added after the substrate is placed in the container. When the absorbent substance is added after the substrate is placed in the container, the absorbent substance can be added from the injection port provided in the container.
（４）で調製した容器に吸光物質を添加する。吸光物質は、乾燥状態であっても液状であってもよい。容器に吸光物質を予め添加しておくことで、蛍光分子で修飾されたターゲット又は捕捉分子を含む溶液を容器に供給した際に、前記溶液に吸光物質を添加することができる。なお、吸光物質の容器への添加は、基板を容器に配置する前に行ってもよく、基板を容器に配置した後でもよい。吸光物質の添加を、基板を容器に配置した後に行う場合は、吸光物質は容器に設けた注入口から添加することができる。 (5) Addition of the absorbent substance to the container Add the absorbent substance to the container prepared in (4). The absorbent material may be in a dry state or in a liquid state. By adding the absorbent substance to the container in advance, the absorbent substance can be added to the solution when the solution containing the target or the capture molecule modified with the fluorescent molecule is supplied to the container. The absorption substance may be added to the container before the substrate is placed in the container, or may be added after the substrate is placed in the container. When the absorbent substance is added after the substrate is placed in the container, the absorbent substance can be added from the injection port provided in the container.
次に、本発明のターゲット計測装置について説明する。
本発明のターゲット計測装置は、本発明のターゲット計測用デバイスと、前記ターゲット計測用デバイスからの、蛍光分子の蛍光量を測定する蛍光読取装置とを有する。
図６は本発明のターゲット計測装置を示す構成図の一例である。本実施形態のターゲット計測装置はターゲット計測用デバイス１０のターゲットと、捕捉分子とが結合する前後の画像を取得するため、結合前の画像を取得後、温調ステージによりターゲット計測用デバイス１０の固相面の温度を上昇させて結合反応を進行させ、再び常温に下げた状態で結合後の画像を取得する。例えば、基板の固相面にＤＮＡプローブ１を固定化する場合は、蛍光分子４で修飾されたターゲット３をＤＮＡプローブ１とハイブリダイズ反応させ、ハイブリダイズ反応前後の画像を取得する。 Next, the target measuring device of the present invention will be described.
The target measuring device of the present invention includes a target measuring device of the present invention and a fluorescence reading device for measuring the amount of fluorescence of a fluorescent molecule from the target measuring device.
FIG. 6 is an example of a configuration diagram showing the target measuring device of the present invention. In the target measuring device of the present embodiment, since the target of thetarget measuring device 10 and the image before and after the binding of the captured molecule are acquired, after the image before the binding is acquired, the target measuring device 10 is fixed by the temperature control stage. The temperature of the phase surface is raised to allow the bonding reaction to proceed, and the image after bonding is acquired in a state where the temperature is lowered to room temperature again. For example, when the DNA probe 1 is immobilized on the solid surface of the substrate, the target 3 modified with the fluorescent molecule 4 is hybridized with the DNA probe 1 to obtain images before and after the hybridization reaction.
本発明のターゲット計測装置は、本発明のターゲット計測用デバイスと、前記ターゲット計測用デバイスからの、蛍光分子の蛍光量を測定する蛍光読取装置とを有する。
図６は本発明のターゲット計測装置を示す構成図の一例である。本実施形態のターゲット計測装置はターゲット計測用デバイス１０のターゲットと、捕捉分子とが結合する前後の画像を取得するため、結合前の画像を取得後、温調ステージによりターゲット計測用デバイス１０の固相面の温度を上昇させて結合反応を進行させ、再び常温に下げた状態で結合後の画像を取得する。例えば、基板の固相面にＤＮＡプローブ１を固定化する場合は、蛍光分子４で修飾されたターゲット３をＤＮＡプローブ１とハイブリダイズ反応させ、ハイブリダイズ反応前後の画像を取得する。 Next, the target measuring device of the present invention will be described.
The target measuring device of the present invention includes a target measuring device of the present invention and a fluorescence reading device for measuring the amount of fluorescence of a fluorescent molecule from the target measuring device.
FIG. 6 is an example of a configuration diagram showing the target measuring device of the present invention. In the target measuring device of the present embodiment, since the target of the
温調ステージは、ターゲットと捕捉分子との結合を促進するために、ターゲットと捕捉分子との反応中に、振とうまたはターゲット計測用デバイスの回転、ボルテックスミキサー等による撹拌機能があることが好ましい。
It is preferable that the temperature control stage has a shaking function, rotation of a target measuring device, stirring function by a vortex mixer, etc. during the reaction between the target and the trapped molecule in order to promote the bond between the target and the trapped molecule.
蛍光読取装置４０の光学系では、レーザー光源４１から出射されたレーザー光はミラー４５を介してダイクロイックミラー４４で反射されターゲット計測用デバイスの固相面を照射する。照射された光がターゲット計測用デバイス１０の固相面上にある蛍光分子４に対する励起光３３となり、蛍光分子４が励起状態になって蛍光分子４が蛍光３４を放射する。
In the optical system of the fluorescent reader 40, the laser light emitted from the laser light source 41 is reflected by the dichroic mirror 44 via the mirror 45 and illuminates the solid phase surface of the target measurement device. The irradiated light becomes the excitation light 33 for the fluorescent molecule 4 on the solid phase surface of the target measurement device 10, the fluorescent molecule 4 is in an excited state, and the fluorescent molecule 4 radiates the fluorescence 34.
ターゲット計測用デバイス１０の固相面から放出された蛍光は、ダイクロイックミラー４４を透過し結像光学系４３を介して、ＣＣＤカメラ４２の検出素子上に蛍光画像が結像し検出される。ここで蛍光３４中に励起光３３が漏れ入るのを防ぐ目的で、励起光３３側に励起光波長に合わせたバンドパスフィルタを設置してもよく、蛍光３４側に検出したい蛍光波長に合わせたバンドパスフィルタを設置してもよい。
The fluorescence emitted from the solid phase surface of the target measurement device 10 passes through the dichroic mirror 44 and is detected by forming a fluorescence image on the detection element of the CCD camera 42 via the imaging optical system 43. Here, in order to prevent the excitation light 33 from leaking into the fluorescence 34, a bandpass filter tuned to the excitation light wavelength may be installed on the excitation light 33 side, or tuned to the fluorescence wavelength to be detected on the fluorescence 34 side. A band pass filter may be installed.
本発明のターゲット計測装置により得られる蛍光画像は、同一スポットのターゲットと捕捉分子の結合前後の画像を取得することができる。そのため、固相間、スポット間の光量のばらつきの影響を受けない。また、結合反応前後の蛍光画像から蛍光変化量を演算し、結合反応した分子数を算出することができる。蛍光変化量の演算は、スポット全体の平均光量を使用してもよいし、スポット画像の各ピクセルの蛍光変化量を使用してもよい。
The fluorescent image obtained by the target measuring device of the present invention can acquire images before and after the binding between the target at the same spot and the captured molecule. Therefore, it is not affected by variations in the amount of light between solid phases and spots. In addition, the amount of change in fluorescence can be calculated from the fluorescence images before and after the binding reaction, and the number of molecules undergoing the binding reaction can be calculated. The calculation of the fluorescence change amount may use the average light amount of the entire spot, or may use the fluorescence change amount of each pixel of the spot image.
本発明のターゲット計測装置は、ＣＣＤカメラ４２をコントロールするコンピュータと、画像の光量を計算する演算装置、画像と光量等を保存する記録装置を備えていてもよい。
The target measuring device of the present invention may include a computer for controlling the CCD camera 42, an arithmetic device for calculating the light amount of the image, and a recording device for storing the image and the light amount.
本発明のターゲット計測装置は、上記実施形態に限定されることはない。本発明のターゲット計測装置は固相面上の検出スポットの固定化面と反対側の面から蛍光を検出するため、蛍光顕微鏡や共焦点顕微鏡、エバネッセント蛍光検出装置、薄膜斜光照明顕微鏡、シート照明顕微鏡、構造化照明顕微鏡、多光子励起顕微鏡などを使用することができる。
The target measuring device of the present invention is not limited to the above embodiment. Since the target measuring device of the present invention detects fluorescence from the surface opposite to the fixed surface of the detection spot on the solid phase surface, a fluorescence microscope, a confocal microscope, an evanescent fluorescence detector, a thin film oblique illumination microscope, and a sheet illumination microscope. , A structured illumination microscope, a multiphoton excitation microscope, etc. can be used.
次に、本発明のターゲット計測用キットについて説明する。本発明のターゲット計測用キットは、本発明のターゲット計測方法に用いることができる。
本発明のターゲット計測用キットは、サンプルに含まれるターゲットを計測する際に用いられるターゲット計測用キットであって、前記ターゲットと前記捕捉分子とのいずれか一方が固相面に固定化された基板と、前記ターゲット及び前記捕捉分子のいずれか他方を含む溶液を、前記基板の前記固相面に接触させた状態で保持可能な容器と、前記ターゲットと前記捕捉分子との結合物を修飾する蛍光分子を励起する励起光、又は前記蛍光分子から発せられる蛍光を吸収する吸光物質と、を含む。 Next, the target measurement kit of the present invention will be described. The target measurement kit of the present invention can be used for the target measurement method of the present invention.
The target measurement kit of the present invention is a target measurement kit used when measuring a target contained in a sample, and is a substrate in which either the target or the capture molecule is immobilized on a solid phase surface. And a container that can hold a solution containing either the target or the capture molecule in contact with the solid phase surface of the substrate, and fluorescence that modifies the conjugate of the target and the capture molecule. It contains an excitation light that excites a molecule or an light-absorbing substance that absorbs the fluorescence emitted from the fluorescent molecule.
本発明のターゲット計測用キットは、サンプルに含まれるターゲットを計測する際に用いられるターゲット計測用キットであって、前記ターゲットと前記捕捉分子とのいずれか一方が固相面に固定化された基板と、前記ターゲット及び前記捕捉分子のいずれか他方を含む溶液を、前記基板の前記固相面に接触させた状態で保持可能な容器と、前記ターゲットと前記捕捉分子との結合物を修飾する蛍光分子を励起する励起光、又は前記蛍光分子から発せられる蛍光を吸収する吸光物質と、を含む。 Next, the target measurement kit of the present invention will be described. The target measurement kit of the present invention can be used for the target measurement method of the present invention.
The target measurement kit of the present invention is a target measurement kit used when measuring a target contained in a sample, and is a substrate in which either the target or the capture molecule is immobilized on a solid phase surface. And a container that can hold a solution containing either the target or the capture molecule in contact with the solid phase surface of the substrate, and fluorescence that modifies the conjugate of the target and the capture molecule. It contains an excitation light that excites a molecule or an light-absorbing substance that absorbs the fluorescence emitted from the fluorescent molecule.
吸光物質は、ターゲット又は捕捉分子を含むサンプル溶液を調製する際にサンプル溶液に添加してもよい。吸光物質をサンプル溶液に添加するタイミングとしては、蛍光分子に励起光を照射する前であればいかなる段階でもよく、例えば、サンプル溶液を調製する段階でも、ターゲットと捕捉分子が固相上で結合する前の段階でもよく、また、ターゲットと捕捉分子が固相上で結合した後の段階であってもよい。
The absorbent material may be added to the sample solution when preparing the sample solution containing the target or capture molecule. The timing of adding the absorbent substance to the sample solution may be any stage as long as it is before the fluorescent molecule is irradiated with the excitation light. For example, even at the stage of preparing the sample solution, the target and the capture molecule are bonded on the solid phase. It may be a previous step, or it may be a step after the target and the capture molecule are bound on the solid phase.
前記容器には予め吸光物質が添加されていてもよい。吸光物質は、乾燥状態であっても液状であってもよい。容器に吸光物質を予め添加しておくことで、蛍光分子で修飾されたターゲット又は捕捉分子を含む溶液を容器に供給した際に、前記溶液に吸光物質を添加することができる。
An absorbent substance may be added to the container in advance. The absorbent material may be in a dry state or in a liquid state. By adding the absorbent substance to the container in advance, the absorbent substance can be added to the solution when the solution containing the target or the capture molecule modified with the fluorescent molecule is supplied to the container.
例えば、前記基板の固相面には、捕捉分子が固定化されており、前記容器は、蛍光分子で修飾されたターゲットと、前記吸光物質とを含む溶液を前記基板の表面に接触させた状態で保持することが可能な容器であってもよく、前記基板の固相面には、ターゲットが固定化されており、前記容器は、蛍光分子で修飾された捕捉分子と、前記吸光物質とを含む溶液を前記固相面に接触させた状態で保持することが可能な容器であってもよい。
For example, a capture molecule is immobilized on the solid phase surface of the substrate, and the container is in a state where a solution containing the target modified with fluorescent molecules and the light-absorbing substance is brought into contact with the surface of the substrate. The target may be immobilized on the solid phase surface of the substrate, and the container may contain a capture molecule modified with a fluorescent molecule and the light-absorbing substance. It may be a container capable of holding the containing solution in contact with the solid phase surface.
また、前記基板の固相面には、蛍光分子で修飾されている捕捉分子が固定化されており、前記容器が、前記ターゲットと前記吸光物質とを含む溶液が前記固相面に接触させた状態で保持することが可能な容器であってもよい。
Further, a capture molecule modified with a fluorescent molecule is immobilized on the solid phase surface of the substrate, and the container is brought into contact with the solid phase surface by a solution containing the target and the absorbent substance. It may be a container that can be held in a state.
また、前記基板の固相面には、前記ターゲット及び前記捕捉分子のいずれか一方が固定化されており、前記容器が、前記ターゲット及び前記捕捉分子のいずれか他方と、前記ターゲットと前記捕捉分子との結合体に結合する蛍光分子と、前記吸光物質と、を含む溶液が前記基板の固相面に接触した状態で保持することが可能な容器であってもよい。
Further, either one of the target and the trapping molecule is immobilized on the solid surface of the substrate, and the container has the target and the trapping molecule on the other side of the target and the trapping molecule. A container may be used in which a solution containing the fluorescent molecule bound to the conjugate of the substrate and the light-absorbing substance can be held in contact with the solid phase surface of the substrate.
ターゲット及び捕捉分子としては、前述のターゲット及び捕捉分子が挙げられる。本発明のターゲット計測用キットとしては、例えば、ターゲットとして、特定の核酸配列を有するターゲットであり、捕捉分子として、前記特定の核酸配列と相補的な配列を有するＤＮＡプローブであるキット等が挙げられる。
Examples of the target and capture molecule include the above-mentioned target and capture molecule. Examples of the target measurement kit of the present invention include a kit having a specific nucleic acid sequence as a target and a DNA probe having a sequence complementary to the specific nucleic acid sequence as a capture molecule. ..
本発明のターゲット計測用キットは、本発明のターゲット計測用デバイスを含むものであってもよい。
The target measurement kit of the present invention may include the target measurement device of the present invention.
本発明のターゲット計測用キットにおいて、基板、吸光物質、容器は前述したものが挙げられる。本発明のターゲット計測用キットは、さらに、ターゲットを定量するために必要な標準液、必要な緩衝液、製品説明書等を含んでいてもよい。
In the target measurement kit of the present invention, the above-mentioned substrates, absorbent substances, and containers can be mentioned. The target measurement kit of the present invention may further include a standard solution necessary for quantifying the target, a necessary buffer solution, a product description, and the like.
次に、本発明のターゲット計測用キットの使用方法を、図４に示したターゲット計測用デバイスを含むキットを例にして説明する。このターゲット計測用キットは、ターゲットが、特定の核酸配列を有するＤＮＡであり、捕捉分子が、前記特定の核酸配列と相補的な配列を有するＤＮＡプローブである。前記ＤＮＡプローブはＤＮＡマイクロアレイの固相面に固定化されており、ターゲットが蛍光分子で修飾されている。
Next, a method of using the target measurement kit of the present invention will be described using a kit including the target measurement device shown in FIG. 4 as an example. In this target measurement kit, the target is DNA having a specific nucleic acid sequence, and the capture molecule is a DNA probe having a sequence complementary to the specific nucleic acid sequence. The DNA probe is immobilized on the solid surface of a DNA microarray, and the target is modified with a fluorescent molecule.
本実施形態のターゲット計測用キットは、ＤＮＡプローブ１が固定化されたＤＮＡマイクロアレイ５と、吸光物質添加ターゲット溶液３５を保持することが可能な容器であって、前記溶液３５を、前記ＤＮＡプローブ１が固定化されているＤＮＡマイクロアレイ５の固相面に接触させた状態で保持している容器３２を含み、容器３２には吸光物質が添加されている。
The target measurement kit of the present embodiment is a container capable of holding a DNA microarray 5 on which a DNA probe 1 is immobilized and a target solution 35 to which an absorbent substance is added, and the solution 35 is used as the DNA probe 1. Includes a container 32 that is held in contact with the solid phase surface of the DNA microarray 5 on which the DNA microarray 5 is immobilized, and an absorbent substance is added to the container 32.
まず、サンプル中のターゲット３を蛍光分子４で修飾し、ターゲット溶液を調製する。ターゲット溶液を調製する際に、特定の核酸配列を有するターゲット３の増幅を行ってもよい。次に、ターゲット溶液を容器３２に供給し、ターゲット溶液を、ＤＮＡマイクロアレイ５の固相表面に接触させる。容器３２に添加されている吸光物質は、ターゲット溶液を容器３２に供給する際に、ターゲット溶液に添加され、吸光物質添加ターゲット溶液３５となる。吸光物質添加ターゲット溶液３５を、ＤＮＡプローブ１が固定化されているＤＮＡマイクロアレイ５の固相面に接触させた後に、蛍光分子４で修飾されたターゲット３と、ＤＮＡマイクロアレイ５に固定化されたＤＮＡプローブ１と、をハイブリダイズ反応させる。このハイブリダイズ反応により、ターゲット３がＤＮＡプローブ１と結合し、ＤＮＡプローブ１が固定化されているＤＮＡスポット３０にターゲット３を修飾している蛍光分子４が捕捉される。
First, the target 3 in the sample is modified with the fluorescent molecule 4 to prepare a target solution. When preparing the target solution, amplification of the target 3 having a specific nucleic acid sequence may be performed. Next, the target solution is supplied to the container 32, and the target solution is brought into contact with the solid phase surface of the DNA microarray 5. The absorbent substance added to the container 32 is added to the target solution when the target solution is supplied to the container 32, and becomes the absorbent substance-added target solution 35. After contacting the target solution 35 to which the absorbent substance is added with the solid phase surface of the DNA microarray 5 on which the DNA probe 1 is immobilized, the target 3 modified with the fluorescent molecule 4 and the DNA immobilized on the DNA microarray 5 The probe 1 and the probe 1 are hybridized. By this hybridization reaction, the target 3 binds to the DNA probe 1, and the fluorescent molecule 4 modifying the target 3 is captured in the DNA spot 30 on which the DNA probe 1 is immobilized.
ハイブリダイズ反応後、蛍光分子４を励起する励起光３３を、ＤＮＡマイクロアレイ５の固相面とは反対側から照射する。
After the hybridization reaction, the excitation light 33 that excites the fluorescent molecule 4 is irradiated from the side opposite to the solid phase surface of the DNA microarray 5.
次に、ＤＮＡマイクロアレイ５に固定化されたＤＮＡプローブ１に結合したターゲット３を修飾する蛍光分子４から発生する蛍光３４を、ＤＮＡマイクロアレイ５の固相面とは反対側から検出する。例えば、蛍光分子４から発生する蛍光画像を蛍光読取装置４０で取得する。そして、取得された蛍光画像から、蛍光量を算出する。
Next, the fluorescence 34 generated from the fluorescent molecule 4 that modifies the target 3 bound to the DNA probe 1 immobilized on the DNA microarray 5 is detected from the side opposite to the solid phase surface of the DNA microarray 5. For example, the fluorescence image generated from the fluorescence molecule 4 is acquired by the fluorescence reader 40. Then, the amount of fluorescence is calculated from the acquired fluorescence image.
本発明の適用範囲は上記実施形態に限定されることはない。本発明は、サンプルに含まれるターゲットを計測するターゲット計測方法、ターゲット計測用デバイス、ターゲット計測装置、及びターゲット計測用キットに対し、広く適用することができる。
The scope of application of the present invention is not limited to the above embodiment. The present invention can be widely applied to a target measurement method for measuring a target contained in a sample, a target measurement device, a target measurement device, and a target measurement kit.
本発明のターゲット計測方法、ターゲット計測用デバイス、ターゲット計測装置、及びターゲット計測用キットは、蛍光分子光量計測におけるドライ画像測定、バイオチップの蛍光分子光量の液中観察、及び連続反応におけるリアルタイム観察等に使用することができる。具体的には、例えば、遺伝子・高分子分析による菌種判別、がん遺伝子、動植物判別、腸内細菌の検査等に用いることができる。
The target measurement method, target measurement device, target measurement device, and target measurement kit of the present invention include dry image measurement in fluorescent molecular light intensity measurement, in-liquid observation of fluorescent molecular light intensity of biochip, and real-time observation in continuous reaction. Can be used for. Specifically, it can be used, for example, for bacterial species discrimination by gene / polymer analysis, oncogene, animal and plant discrimination, intestinal bacterial test, and the like.
また、本発明のターゲット計測方法、ターゲット計測用デバイス、ターゲット計測装置、及びターゲット計測用キットは、臨床検査等に使用される標識抗体法等のような固相法にも適用される。例えば、組織や細胞内の特定の染色体や遺伝子の発現を、蛍光物質を用いて蛍光測定するＦＩＳＨ法（蛍光 ｉｎ ｓｉｔｕ ハイブリダイゼーション）が一例として挙げられる。この他にも、ユーロピウム等の蛍光発光物質を標識として抗原抗体反応を測定するＦＩＡ法（蛍光免疫測定法）や、抗原となる病原体等に蛍光物質をラベルした血清（抗体）反応を測定するＩＦＡ法（間接蛍光抗体法）にも適用される。
Further, the target measurement method, the target measurement device, the target measurement device, and the target measurement kit of the present invention are also applied to a solid phase method such as a labeled antibody method used for clinical examinations and the like. For example, the FISH method (fluorescence in situ hybridization) in which the expression of a specific chromosome or gene in a tissue or cell is measured by fluorescence using a fluorescent substance can be mentioned as an example. In addition to this, the FIA method (fluorescence immunofluorescence measurement method) that measures the antigen-antibody reaction using a fluorescent luminescent substance such as europium as a label, and the IFA that measures the serum (antibody) reaction that labels the fluorescent substance on the pathogen that becomes the antigen. It also applies to the method (indirect fluorescent antibody method).
以下、実施例に基づき本発明を更に詳細に説明するが、本発明はこれらにより限定されるものではない。
Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited thereto.
（実施例１）
吸光物質添加による背景光の低減効果を確認した。容器内に蛍光分子４としてＣｙ３（登録商標）分子を０．３から３，０００ｎＭの濃度になるように調整し、容器に対して透明ガラス基板を配置した。透明ガラス基板を透過して５３２ｎｍの励起光３３を照射し、蛍光画像を取得した。
図７に、容器中の溶液に吸光物質を添加した場合と、添加しなかった場合とで、取得した蛍光画像から背景光の光量を算出した結果を示す。また、表１に、蛍光分子の濃度を変えたときの、背景光比（吸光物質を添加しなかったときの背景光の光量／吸光物質を添加したときの背景光の光量）を示す。なお、吸光物質はデキストランコートされた酸化鉄（Ｆｅ２Ｏ３）の粒径５０ｎｍのものを用い、溶液中で５０ｍｇ／ｍｌになるように添加した。 (Example 1)
The effect of reducing the background light by adding the absorbent substance was confirmed. Cy3® molecules were adjusted to a concentration of 0.3 to 3,000 nM asfluorescent molecules 4 in the container, and a transparent glass substrate was placed on the container. A fluorescent image was acquired by transmitting the excitation light 33 at 532 nm through the transparent glass substrate.
FIG. 7 shows the results of calculating the amount of background light from the acquired fluorescent image when the absorbent substance was added to the solution in the container and when it was not added. In addition, Table 1 shows the background light ratio (the amount of background light when the absorbent substance is not added / the amount of background light when the absorbent substance is added) when the concentration of the fluorescent molecule is changed. The light-absorbing substance used was dextran-coated iron oxide (Fe 2 O 3 ) having a particle size of 50 nm, and was added so as to be 50 mg / ml in the solution.
吸光物質添加による背景光の低減効果を確認した。容器内に蛍光分子４としてＣｙ３（登録商標）分子を０．３から３，０００ｎＭの濃度になるように調整し、容器に対して透明ガラス基板を配置した。透明ガラス基板を透過して５３２ｎｍの励起光３３を照射し、蛍光画像を取得した。
図７に、容器中の溶液に吸光物質を添加した場合と、添加しなかった場合とで、取得した蛍光画像から背景光の光量を算出した結果を示す。また、表１に、蛍光分子の濃度を変えたときの、背景光比（吸光物質を添加しなかったときの背景光の光量／吸光物質を添加したときの背景光の光量）を示す。なお、吸光物質はデキストランコートされた酸化鉄（Ｆｅ２Ｏ３）の粒径５０ｎｍのものを用い、溶液中で５０ｍｇ／ｍｌになるように添加した。 (Example 1)
The effect of reducing the background light by adding the absorbent substance was confirmed. Cy3® molecules were adjusted to a concentration of 0.3 to 3,000 nM as
FIG. 7 shows the results of calculating the amount of background light from the acquired fluorescent image when the absorbent substance was added to the solution in the container and when it was not added. In addition, Table 1 shows the background light ratio (the amount of background light when the absorbent substance is not added / the amount of background light when the absorbent substance is added) when the concentration of the fluorescent molecule is changed. The light-absorbing substance used was dextran-coated iron oxide (Fe 2 O 3 ) having a particle size of 50 nm, and was added so as to be 50 mg / ml in the solution.
図７及び表１に示したように、Ｃｙ３分子濃度によって１／４から１／１７に背景光が低減できており、背景光が高い場合に低減効果が高くなっている。しかし、微生物からゲノムＤＮＡを抽出した際、溶液の蛍光が１０μＷ／ｍ２程度存在することが分かっており、背景光が１０μＷ／ｍ２程度の溶液においても効果が出ていることから、サンプルから持ち込まれたターゲット３以外の分子などにより起こる溶液を励起することによって発生する蛍光についても効果があることが分かった。またＣｙ３分子を含まない水の状態においても背景光を１／２に低減できているが、これは溶液中を励起光３３が透過しなくなったことにより、容器底面の反射や自家蛍光が低下したことによるものと考えられ、副次的な効果が確認された。
As shown in FIGS. 7 and 1, the background light can be reduced from 1/4 to 1/17 depending on the Cy3 molecular concentration, and the reduction effect is high when the background light is high. However, when the genomic DNA is extracted from the microorganism, it is known that the fluorescence of the solution is about 10 μW / m 2 , and the effect is obtained even in the solution with the background light of about 10 μW / m 2 , from the sample. It was found that the fluorescence generated by exciting the solution generated by molecules other than the brought-in target 3 is also effective. In addition, the background light can be reduced to 1/2 even in the state of water containing no Cy3 molecule, but this is because the excitation light 33 does not pass through the solution, so that the reflection on the bottom surface of the container and the autofluorescence are reduced. It is considered that this is the cause, and a secondary effect was confirmed.
また、背景光を低減したことによるスポットの見え方の違いを検討した。図８は、Ｃｙ３分子修飾した合成ＤＮＡを基板上に固定化したスポットのＣｙ３分子濃度３から３００ｎＭの溶液中での蛍光読取装置による露光時間１秒のスポット画像である。Ｃｙ３分子濃度が３０ｎＭ以上の場合、１／１０以上に背景光を低減できることから、溶液が呈する蛍光が高い状態でもスポット観察が可能であることが分かった。
We also examined the difference in the appearance of spots due to the reduction of background light. FIG. 8 is a spot image of a spot in which a synthetic DNA modified with Cy3 molecules is immobilized on a substrate and has an exposure time of 1 second by a fluorescence reader in a solution having a Cy3 molecule concentration of 3 to 300 nM. It was found that when the Cy3 molecular concentration is 30 nM or more, the background light can be reduced to 1/10 or more, so that spot observation is possible even when the fluorescence exhibited by the solution is high.
Ｃｙ３分子濃度が３０ｎＭの時のスポット光量と背景光の関係を図９に示す。スポットの光量と背景光の差からスポットのＤＮＡプローブ１に捕捉されている蛍光分子４による蛍光量が算出されるが、図９に示したように、吸光物質添加の有無で光量が変化しておらず、吸光物質を添加することにより検出信号が低下することがなかった。また、スポット光量と背景光の比をＳ／Ｎとすると、吸光物質を添加しなかった時のＳ／Ｎが１．３であるのに対して、吸光物質を添加した時はＳ／Ｎが５．３となり、Ｓ／Ｎが４．２倍向上していた。
FIG. 9 shows the relationship between the amount of spot light and the background light when the Cy3 molecular concentration is 30 nM. The amount of fluorescence due to the fluorescent molecule 4 captured by the DNA probe 1 of the spot is calculated from the difference between the amount of light of the spot and the background light. No, the detection signal was not lowered by adding the absorbent substance. When the ratio of the amount of spot light to the background light is S / N, the S / N is 1.3 when the absorbent substance is not added, whereas the S / N is when the absorbent substance is added. It became 5.3, and the S / N was improved by 4.2 times.
（実施例２）
蛍光分子４非修飾のＤＮＡプローブ１を基板上に複数配置したＤＮＡマイクロアレイ５を作製し、Ｃｙ３分子を修飾したターゲットＤＮＡのハイブリダイズによるスポット観察において吸光物質添加の適用性を以下のようにして確認した。
容器に０．２５ｎＭになるようにＣｙ３分子で修飾されたターゲットＤＮＡを調製し、このとき実施例１と同じ条件で吸光物質を添加し、ＤＮＡマイクロアレイ５を配置した。６０℃、５ｒｐｍで、３０分間インキュベートし、ＤＮＡプローブ１とターゲットＤＮＡをハイブリダイズ反応させた。常温に戻した後、蛍光読取装置によりスポットの蛍光画像を取得し、光量を算出した。その結果を図１０に示す。 (Example 2)
ADNA microarray 5 in which a plurality of unmodified DNA probes 1 were arranged on a substrate was prepared, and the applicability of the addition of an absorbent substance was confirmed in spot observation by hybridization of a target DNA modified with a Cy3 molecule as follows. did.
A target DNA modified with a Cy3 molecule so as to be 0.25 nM was prepared in a container, and at this time, an absorbent substance was added under the same conditions as in Example 1, and aDNA microarray 5 was placed. Incubation was carried out at 60 ° C. and 5 rpm for 30 minutes to hybridize the DNA probe 1 and the target DNA. After returning to room temperature, a fluorescence image of the spot was acquired by a fluorescence reader, and the amount of light was calculated. The results are shown in FIG.
蛍光分子４非修飾のＤＮＡプローブ１を基板上に複数配置したＤＮＡマイクロアレイ５を作製し、Ｃｙ３分子を修飾したターゲットＤＮＡのハイブリダイズによるスポット観察において吸光物質添加の適用性を以下のようにして確認した。
容器に０．２５ｎＭになるようにＣｙ３分子で修飾されたターゲットＤＮＡを調製し、このとき実施例１と同じ条件で吸光物質を添加し、ＤＮＡマイクロアレイ５を配置した。６０℃、５ｒｐｍで、３０分間インキュベートし、ＤＮＡプローブ１とターゲットＤＮＡをハイブリダイズ反応させた。常温に戻した後、蛍光読取装置によりスポットの蛍光画像を取得し、光量を算出した。その結果を図１０に示す。 (Example 2)
A
A target DNA modified with a Cy3 molecule so as to be 0.25 nM was prepared in a container, and at this time, an absorbent substance was added under the same conditions as in Example 1, and a
図１０に示したように、吸光物質を添加した場合は、溶液中に遊離した未反応のＣｙ３分子で修飾されたターゲットの蛍光が低減し、背景光が１／３．７になっていた。このとき、スポット光量と背景光の比をＳ／Ｎとすると、吸光物質を添加しなかった時のＳ／Ｎが１．５であるのに対して、吸光物質を添加した時のＳ／Ｎは３．７となり、Ｓ／Ｎが２．５倍向上していた。吸光物質を添加しなかった時と比較して、吸光物質を添加した時のスポット光量と背景光の差で光量低下が起こっていないことから、吸光物質がＤＮＡプローブ１とターゲット３のハイブリダイズ反応の阻害をしていないことが分かる。
As shown in FIG. 10, when the absorbent substance was added, the fluorescence of the target modified with the unreacted Cy3 molecule released in the solution was reduced, and the background light was reduced to 1 / 3.7. At this time, assuming that the ratio of the amount of spot light to the background light is S / N, the S / N when the absorbent substance is not added is 1.5, whereas the S / N when the absorbent substance is added is 1.5. Was 3.7, and the S / N was improved 2.5 times. Compared with the case where the absorbent substance was not added, the light intensity did not decrease due to the difference between the spot light amount and the background light when the absorbent substance was added. Therefore, the absorbent substance is the hybrid reaction between the DNA probe 1 and the target 3. It can be seen that it does not inhibit.
１ ＤＮＡプローブ
２ 検出配列
３ ターゲット
４ 蛍光分子
５ ＤＮＡマイクロアレイ
６ ドナー蛍光プローブ
７ 消光プローブ
８ 消光物質
１０ ターゲット計測用デバイス
３０ ＤＮＡスポット
３２ 容器
３３ 励起光
３４ 蛍光
３５ 吸光物質添加ターゲット溶液
４０ 蛍光読取装置
４１ レーザー光源
４２ ＣＣＤカメラ
４３ 結像光学系
４４ ダイクロイックミラー
４５ ミラー
４６ ステージ 1DNA probe 2 Detection sequence 3 Target 4 Fluorescent molecule 5 DNA microarray 6 Donor fluorescent probe 7 Quenching probe 8 Quenching substance 10 Target measurement device 30 DNA spot 32 Container 33 Excitation light 34 Fluorescent 35 Absorbent-added target solution 40 Fluorescent reader 41 Laser light source 42 CCD camera 43 Imaging optical system 44 Dichromic mirror 45 Mirror 46 Stage
２ 検出配列
３ ターゲット
４ 蛍光分子
５ ＤＮＡマイクロアレイ
６ ドナー蛍光プローブ
７ 消光プローブ
８ 消光物質
１０ ターゲット計測用デバイス
３０ ＤＮＡスポット
３２ 容器
３３ 励起光
３４ 蛍光
３５ 吸光物質添加ターゲット溶液
４０ 蛍光読取装置
４１ レーザー光源
４２ ＣＣＤカメラ
４３ 結像光学系
４４ ダイクロイックミラー
４５ ミラー
４６ ステージ 1
Claims (20)
- サンプルに含まれるターゲットを計測するターゲット計測方法であって、
蛍光分子で修飾された、前記ターゲットと前記ターゲットに特異的に結合する捕捉分子との結合物が設けられた基板の固相面に、前記蛍光分子を励起する励起光、又は前記蛍光分子から発せられる蛍光を吸収する吸光物質を含む溶液が接触している状態で、前記基板の前記固相面とは反対側から前記励起光を照射して得られる蛍光を、前記基板の前記固相面とは反対側から計測するターゲット計測方法。 It is a target measurement method that measures the target contained in the sample.
The excitation light that excites the fluorescent molecule or the fluorescent molecule emits light on the solid phase surface of the substrate provided with the conjugate of the target and the capture molecule that specifically binds to the target, which is modified with the fluorescent molecule. The fluorescence obtained by irradiating the excitation light from the side opposite to the solid phase surface of the substrate in a state where the solution containing the absorbent substance that absorbs the fluorescence is in contact with the solid phase surface of the substrate is applied to the solid surface surface of the substrate. Is a target measurement method that measures from the opposite side. - 前記ターゲットを前記蛍光分子で修飾し、
前記固相面に固定化されている前記捕捉分子に前記ターゲットを結合させて前記結合物を得る、請求項１に記載のターゲット計測方法。 The target is modified with the fluorescent molecule and
The target measurement method according to claim 1, wherein the target is bound to the capture molecule immobilized on the solid phase surface to obtain the conjugate. - 前記捕捉分子を前記蛍光分子で修飾し、
前記固相面に固定化されている前記ターゲットに前記捕捉分子を結合させて前記結合物を得る、請求項１に記載のターゲット計測方法。 The capture molecule is modified with the fluorescent molecule and
The target measurement method according to claim 1, wherein the capture molecule is bound to the target immobilized on the solid phase surface to obtain the conjugate. - 前記捕捉分子を前記蛍光分子で修飾し、
前記固相面に固定化されている前記捕捉分子に、前記ターゲットを結合させて前記結合物を得る、請求項１に記載のターゲット計測方法。 The capture molecule is modified with the fluorescent molecule and
The target measurement method according to claim 1, wherein the target is bound to the trapping molecule immobilized on the solid phase surface to obtain the conjugate. - 前記固相面に固定化されている、前記ターゲット及び前記捕捉分子のいずれか一方に対し、前記ターゲット及び前記捕捉分子のいずれか他方と前記蛍光分子とを供給して前記結合物を得る、請求項１に記載のターゲット計測方法。 Claimed to obtain the conjugate by supplying either the target or the capture molecule and the fluorescent molecule to either the target or the capture molecule immobilized on the solid phase surface. Item 1. The target measurement method according to Item 1.
- 前記ターゲットは、特定の核酸配列を有する核酸であり、
前記捕捉分子は、前記特定の核酸配列と相補的な配列を有する検出プローブであり、
前記ターゲットを前記検出プローブにハイブリダイズ反応させて前記結合物を得る、請求項１～５のいずれか１項に記載のターゲット計測方法。 The target is a nucleic acid having a specific nucleic acid sequence.
The capture molecule is a detection probe having a sequence complementary to the specific nucleic acid sequence.
The target measurement method according to any one of claims 1 to 5, wherein the target is hybridized with the detection probe to obtain the conjugate. - サンプルに含まれるターゲットを計測する際に用いられるターゲット計測用デバイスであって、
前記ターゲットと前記ターゲットに特異的に結合する捕捉分子とのいずれか一方が固相面に固定化される基板と、
前記ターゲットと前記捕捉分子との結合物を修飾する蛍光分子を励起する励起光、又は前記蛍光分子から発せられる蛍光を吸収する吸光物質が添加された容器であって、前記ターゲット及び前記捕捉分子のいずれか他方と、前記吸光物質とを含む溶液を、前記基板の前記固相面に接触させた状態で保持可能な容器と、
を備えたターゲット計測用デバイス。 A target measurement device used to measure the target contained in the sample.
A substrate in which either one of the target and a capture molecule specifically bound to the target is immobilized on a solid phase surface, and a substrate.
A container to which an excitation light that excites a fluorescent molecule that modifies a bond between the target and the capture molecule or an absorbent substance that absorbs the fluorescence emitted from the fluorescent molecule is added, that is, the target and the capture molecule. A container capable of holding a solution containing either the other and the absorbent substance in contact with the solid phase surface of the substrate.
A device for target measurement equipped with. - 前記固相面には、前記捕捉分子が固定化されており、
前記容器には、前記蛍光分子で修飾された前記ターゲットを含む溶液が供給される、請求項７に記載のターゲット計測用デバイス。 The capture molecule is immobilized on the solid phase surface, and the capture molecule is immobilized on the solid surface.
The target measurement device according to claim 7, wherein the container is supplied with a solution containing the target modified with the fluorescent molecule. - 前記固相面には、前記ターゲットが固定化されており、
前記容器には、前記蛍光分子で修飾された前記捕捉分子を含む溶液が供給される、請求項７に記載のターゲット計測用デバイス。 The target is immobilized on the solid surface.
The target measurement device according to claim 7, wherein the container is supplied with a solution containing the capture molecule modified with the fluorescent molecule. - 前記固相面には、前記蛍光分子で修飾されている、前記捕捉分子が固定化されており、
前記容器には、前記ターゲットを含む溶液が供給される、請求項７に記載のターゲット計測用デバイス。 The capture molecule modified with the fluorescent molecule is immobilized on the solid phase surface.
The target measurement device according to claim 7, wherein a solution containing the target is supplied to the container. - 前記固相面には、前記ターゲット及び前記捕捉分子のいずれか一方が固定化されており、
前記容器には、前記ターゲット及び前記捕捉分子のいずれか他方と、前記ターゲットと前記捕捉分子との結合体に結合する前記蛍光分子と、を含む溶液が供給される、請求項７に記載のターゲット計測用デバイス。 Either the target or the capture molecule is immobilized on the solid phase surface.
The target according to claim 7, wherein the container is supplied with a solution containing any one of the target and the capture molecule and the fluorescent molecule that binds to the conjugate of the target and the capture molecule. Measurement device. - 前記ターゲットは、特定の核酸配列を有するターゲットであり、
前記捕捉分子は、前記特定の核酸配列に相補的な配列を有する検出プローブである、請求項７～１１のいずれか１項に記載のターゲット計測用デバイス。 The target is a target having a specific nucleic acid sequence.
The target measurement device according to any one of claims 7 to 11, wherein the capture molecule is a detection probe having a sequence complementary to the specific nucleic acid sequence. - 請求項７～１２のいずれか１項に記載のターゲット計測用デバイスと、
前記ターゲット計測用デバイスからの蛍光量を測定する蛍光読取装置と、
を有する、ターゲット計測装置。 The target measurement device according to any one of claims 7 to 12, and the target measurement device.
A fluorescence reader that measures the amount of fluorescence from the target measurement device,
Has a target measuring device. - サンプルに含まれるターゲットを計測する際に用いられるターゲット計測用キットであって、
前記ターゲットと前記ターゲットに特異的に結合する捕捉分子とのいずれか一方が固相面に固定化された基板と、
前記ターゲット及び前記捕捉分子のいずれか他方を含む溶液を、前記基板の前記固相面に接触させた状態で保持可能な容器と、
前記ターゲットと前記捕捉分子との結合物を修飾する蛍光分子を励起する励起光、又は前記蛍光分子から発せられる蛍光を吸収する吸光物質と、
を含むターゲット計測用キット。 It is a target measurement kit used when measuring the target included in the sample.
A substrate in which one of the target and a capture molecule that specifically binds to the target is immobilized on a solid phase surface, and a substrate.
A container capable of holding a solution containing either one of the target and the trapping molecule in contact with the solid phase surface of the substrate.
Excitation light that excites a fluorescent molecule that modifies the conjugate of the target and the capture molecule, or an absorbent substance that absorbs the fluorescence emitted from the fluorescent molecule.
Target measurement kit including. - 前記吸光物質は、前記容器に予め添加されている、請求項１４に記載のターゲット計測用キット。 The target measurement kit according to claim 14, wherein the absorbent substance is preliminarily added to the container.
- 前記固相面には、前記捕捉分子が固定化されており、
前記容器には、前記蛍光分子で修飾された前記ターゲットと、前記吸光物質とを含む溶液が保持される、請求項１４又は１５に記載のターゲット計測用キット。 The capture molecule is immobilized on the solid phase surface, and the capture molecule is immobilized on the solid surface.
The target measurement kit according to claim 14 or 15, wherein the container holds a solution containing the target modified with the fluorescent molecule and the absorbent substance. - 前記固相面には、前記ターゲットが固定化されており、
前記容器には、前記蛍光分子で修飾された前記捕捉分子と、前記吸光物質とを含む溶液が保持される、請求項１４又は１５に記載のターゲット計測用キット。 The target is immobilized on the solid surface.
The target measurement kit according to claim 14 or 15, wherein the container holds a solution containing the capture molecule modified with the fluorescent molecule and the absorbent substance. - 前記固相面には、前記蛍光分子で修飾されている前記捕捉分子が固定化されており、
前記容器には、前記ターゲットと前記吸光物質とを含む溶液が保持される、請求項１４又は１５に記載のターゲット計測用キット。 The capture molecule modified with the fluorescent molecule is immobilized on the solid phase surface.
The target measurement kit according to claim 14 or 15, wherein a solution containing the target and the absorbent substance is held in the container. - 前記固相面には、前記ターゲット及び前記捕捉分子のいずれか一方が固定化されており、
前記容器には、前記ターゲット及び前記捕捉分子のいずれか他方と、前記ターゲットと前記捕捉分子との結合体に結合する前記蛍光分子と、前記吸光物質と、を含む溶液が保持される、請求項１４又は１５に記載のターゲット計測用キット。 Either the target or the capture molecule is immobilized on the solid phase surface.
The container holds a solution containing either one of the target and the capture molecule, the fluorescent molecule that binds to the conjugate of the target and the capture molecule, and the absorbent substance. The target measurement kit according to 14 or 15. - 前記ターゲットは、特定の核酸配列を有するターゲットであり、
前記捕捉分子は、前記特定の核酸配列に相補的な配列を有する検出プローブである、請求項１４～１９のいずれか１項に記載のターゲット計測用キット。 The target is a target having a specific nucleic acid sequence.
The target measurement kit according to any one of claims 14 to 19, wherein the capture molecule is a detection probe having a sequence complementary to the specific nucleic acid sequence.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202180051930.8A CN116171319A (en) | 2020-08-31 | 2021-07-30 | Target detection method, target detection device, target detection apparatus, and target detection kit |
| JP2022545580A JPWO2022044702A1 (en) | 2020-08-31 | 2021-07-30 | |
| EP21861130.9A EP4206332A4 (en) | 2020-08-31 | 2021-07-30 | Target measurement method, device for target measurement, target measurement apparatus, and kit for target measurement |
| US18/023,249 US20230314419A1 (en) | 2020-08-31 | 2021-07-30 | Target measurement method, target measurement device, target measurement apparatus, and target measurement kit |
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| JP2020-145840 | 2020-08-31 | ||
| JP2020145840 | 2020-08-31 |
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| PCT/JP2021/028455 WO2022044702A1 (en) | 2020-08-31 | 2021-07-30 | Target measurement method, device for target measurement, target measurement apparatus, and kit for target measurement |
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| Country | Link |
|---|---|
| US (1) | US20230314419A1 (en) |
| EP (1) | EP4206332A4 (en) |
| JP (1) | JPWO2022044702A1 (en) |
| CN (1) | CN116171319A (en) |
| WO (1) | WO2022044702A1 (en) |
Citations (5)
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|---|---|---|---|---|
| JPH07120397A (en) * | 1993-08-31 | 1995-05-12 | Daikin Ind Ltd | Apparatus and method for optical measurement |
| JP2001194310A (en) * | 2000-01-17 | 2001-07-19 | Yokogawa Electric Corp | Device for reading biochip |
| JP2001311690A (en) * | 2000-04-28 | 2001-11-09 | Yokogawa Electric Corp | Biochip reader and electrophoretic apparatus |
| JP2003084002A (en) * | 2001-06-27 | 2003-03-19 | Fuji Photo Film Co Ltd | Method for reducing background in fluorescence detection |
| JP2015043702A (en) | 2013-08-27 | 2015-03-12 | 横河電機株式会社 | Method for measuring nucleic acid sequence, device for measuring nucleic acid sequence, producing method of device for measuring nucleic acid sequence and apparatus for measuring nucleic acid sequence |
-
2021
- 2021-07-30 EP EP21861130.9A patent/EP4206332A4/en active Pending
- 2021-07-30 WO PCT/JP2021/028455 patent/WO2022044702A1/en unknown
- 2021-07-30 US US18/023,249 patent/US20230314419A1/en active Pending
- 2021-07-30 JP JP2022545580A patent/JPWO2022044702A1/ja active Pending
- 2021-07-30 CN CN202180051930.8A patent/CN116171319A/en active Pending
Patent Citations (5)
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|---|---|---|---|---|
| JPH07120397A (en) * | 1993-08-31 | 1995-05-12 | Daikin Ind Ltd | Apparatus and method for optical measurement |
| JP2001194310A (en) * | 2000-01-17 | 2001-07-19 | Yokogawa Electric Corp | Device for reading biochip |
| JP2001311690A (en) * | 2000-04-28 | 2001-11-09 | Yokogawa Electric Corp | Biochip reader and electrophoretic apparatus |
| JP2003084002A (en) * | 2001-06-27 | 2003-03-19 | Fuji Photo Film Co Ltd | Method for reducing background in fluorescence detection |
| JP2015043702A (en) | 2013-08-27 | 2015-03-12 | 横河電機株式会社 | Method for measuring nucleic acid sequence, device for measuring nucleic acid sequence, producing method of device for measuring nucleic acid sequence and apparatus for measuring nucleic acid sequence |
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| See also references of EP4206332A4 |
| VIVIAN G. C. ET AL.: "Making and reading microarrays", NATURE GENETICS SUPPLEMENT, vol. 21, 1999, pages 15 - 19 |
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| Publication number | Publication date |
|---|---|
| EP4206332A4 (en) | 2024-10-09 |
| EP4206332A1 (en) | 2023-07-05 |
| US20230314419A1 (en) | 2023-10-05 |
| CN116171319A (en) | 2023-05-26 |
| JPWO2022044702A1 (en) | 2022-03-03 |
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